Tau PET imaging ligands

ABSTRACT

The present invention relates to novel, selective radiolabelled tau ligands which are useful for imaging and quantifying tau aggregates, using positron-emission tomography (PET). The invention is also directed to compositions comprising such compounds, to processes for preparing such compounds and compositions, to the use of such compounds and compositions for imaging a tissue or a subject, in vitro or in vivo, and to precursors of said compounds.

This application is a 371 National Stage Application of InternationalApplication No. PCT/EP2017/067898 with an international filing date ofJul. 14, 2017, which claims the benefit of European Application No.EP17152062.0 filed Jan. 1, 2017, European Application No. EP16204242.8filed Dec. 15, 2016 and Provisional Application No. 62/363,452 filingdate of Jul. 18, 2016 the entire disclosures of each of which are herebyincorporated in their entirety.

FIELD OF THE INVENTION

The present invention relates to novel, selective radiolabelled tauligands which are useful for imaging and quantifying tau aggregates,using positron-emission tomography (PET). The invention is also directedto compositions comprising such compounds, to processes for preparingsuch compounds and compositions, to the use of such compounds andcompositions for imaging a tissue or a subject, in vitro or in vivo, andto precursors of said compounds.

BACKGROUND OF THE INVENTION

Alzheimer's Disease (AD) is a neurodegenerative disease associated withaging. AD patients suffer from cognition deficits and memory loss aswell as behavioural problems such as anxiety. Over 90% of thoseafflicted with AD have a sporadic form of the disorder while less than10% of the cases are familial or hereditary. In the United States, aboutone in ten people at age 65 have AD while at age 85, one out of everytwo individuals are afflicted by AD. The average life expectancy fromthe initial diagnosis is 7-10 years, and AD patients require extensivecare either in an assisted living facility or by family members. Withthe increasing number of elderly in the population, AD is a growingmedical concern. Currently available therapies for AD merely treat thesymptoms of the disease but not the underlying pathology causing thedisease.

The hallmark pathological features in the brain of AD patients areneurofibrillary tangles which are generated by aggregates ofhyperphosphorylated tau protein and amyloid plaques which form byaggregation of beta-amyloid peptide.

Though the most prevalent neurodegenerative disorder is AD, aggregatedtau protein is also a characteristic of other neurodegenerative diseasesknown as “tauopathies”, which additionally but not exclusively includetangle-only dementia (TD), argyrophilic grain disease (AGD), progressivesupranuclear palsy (PSP), corticobasal degeneration (CBD), Pick disease(PiD), and frontotemporal dementia and parkinsonism linked to chromosome17 (FTDP-17). The heterogeneity of these disorders is closely related tothe wide range of human tau isoforms and post-translationalmodifications. Tau aggregates may appear ultrastructurally as pairedhelical filaments (PHF), straight filaments (SF), randomly coiledfilaments (RCF), or twisted filaments (TF); this variability translatesinto polymorphism. A correlation of neurofibrillary tangles has beenmade with the level of cognitive impairment in AD and/or the chance ofdeveloping AD. However, diagnosis can still only be performedpost-mortem by means of biopsy/autopsy. Examination based on history andstatistical memory testing require clear evidence of impairment ordementia, and are often inaccurate or insensitive, and measurement of Aβpeptides and total tau proteins in cerebrospinal fluid by lumbarpuncture is invasive and amenable to adverse effects. Apart from theintrinsic complexity of AD, the development of a cure has been hamperedby the lack of reliable tools for early diagnosis, staging, andaccurately monitoring disease progression. There is therefore still aneed to identify a means to perform diagnosis and/or monitor diseaseprogression. Imaging of tau aggregates may provide such means,particularly when anti-tau treatments emerge.

Positron Emission Tomography (PET) is a non-invasive imaging techniquethat offers the highest spatial and temporal resolution of all nuclearimaging techniques and has the added advantage that it can allow fortrue quantification of tracer concentrations in tissues. It usespositron emitting radionuclides for detection.

Several positron emission tomography radiotracers have been reported sofar for imaging of tau aggregates (for a review, see for instance Arizaet al. J. Med. Chem. 2015, 58, 4365-4382). “Preclinical Characterizationof ¹⁸F-MK-6240, a promising PET Tracer for In Vivo Quantification ofHuman Neurofibrillary Tangles” in J. Nucl. Med 2016; 57: 1599-1610disclose 6-fluoro-3-(1H-pyrrolo[2,3-c]pyridine-1-yl)isoquinoline-5-aminewhich binds with high affinity to human AD brain cortex homogenatescontaining abundant NFT but binds poorly to amyloid plaque-rich,NFT-poor AD brain homogenates. Moderate defluorination is observed with¹⁸F-MK-6240 as skull uptake. The article “Radiopharmaceuticals forpositron emission tomography investigations of Alzheimer's disease” inEur. J. Nucl. Med. Imaging 2010; 37(8): 1575-1593 reports on anisoquinoline compound [¹¹C]PK11195 which binds to peripheral corticalbenzodiazepine receptors in activated microglia and which was furtherreported in “Carbon 11-labeled Pittsburgh compound B and carbon11-labeled (R)-PK11195 positron emission tomographic imaging inAlzheimer's disease in Arch. Neurol. 2009; 66(1): 60-67 as failing toshow any differences in brain retention between patients and healthyvolunteers. PET imaging of the human brain with the quinoline compound¹⁸F-THK5351 having high affinity to PHF was shown to be confounded byoff-target binding to the enzyme monoamine oxidase MAO-B (Alzheimer'sResearch & Therapy 2017; 9; 25 DOI 10.1186/s13195-017-0253-y).

There is still a need to provide selective, improved positron emissiontomography radiotracers for imaging tau aggregates with a good balanceof properties including, but not limited to, high affinity andselectivity towards tau aggregates, reversible binding, permeability,suitable brain pharmacokinetic profile, i.e. rapid distributionthroughout the brain, rapid clearance, minimal non-specific binding, andsynthetic accessibility.

SUMMARY OF THE INVENTION

It is therefore an object of the present invention to provideisoquinoline-6-amine compounds useful as tau PET radiotracers.Therefore, in one aspect, the present invention relates to a compoundhaving the Formula (I)

whereinat least one atom is radioactive, and whereinR² is methyl, and either R¹ is F and R³ is H, or R¹ is H and R³ is F; orR¹ and R³ are both H, and R² is selected from the group consisting of—C₁₋₄alkyl-F, —OC₁₋₄alkyl-F, and —NR⁴—C₁₋₄alkyl-F, wherein R⁴ is H ormethyl;or a pharmaceutically acceptable salt or a solvate thereof.

In particular, the present invention relates to a compound of Formula(I′)

whereinR² is methyl, and either R¹ is ¹⁸F and R³ is H, or R¹ is H and R³ is¹⁸F; orR¹ and R³ are both H, and R² is selected from the group consisting of—C₁₋₄alkyl-¹⁸F, —OC₁₋₄alkyl-¹⁸F, and —NR⁴—C₁₋₄alkyl-¹⁸F, wherein R⁴ is Hor methyl;or a pharmaceutically acceptable salt or a solvate thereof.

In another aspect, the invention relates to precursor compounds for thesynthesis of the compounds of Formula (I) or (I′), as previouslydefined. Thus, the present invention also relates to a compound ofFormula (P-1)

whereinR² is methyl, and either R¹ is selected from the group consisting of Br,—NO₂, —[N(CH₃)₃]⁺, and 4-CH₃-Ph-SO₂—O—, and R³ is H, or R¹ is H and R³is selected from the group consisting of Br, —NO₂, —[N(CH₃)₃]⁺, and4-CH₃-Ph-SO₂—O—; orR¹ and R³ are both H, and R² is selected from the group consisting of—C₁₋₄alkyl-Br, —C₁₋₄alkyl-I, —C₁₋₄alkyl-O—SO₂CH₃,4-CH₃-Ph-SO₂—O—C₁₋₄alkyl-, —C₁₋₄alkyl-OH, —OC₁₋₄alkyl-Br, —OC₁₋₄alkyl-I,—OC₁₋₄alkyl-O—SO₂CH₃, 4-CH₃-Ph-SO₂—O—C₁₋₄alkyl-O—, —OC₁₋₄alkyl-OH,—NR⁴—C₁₋₄alkyl-Br, —NR⁴—C₁₋₄alkyl-I, —NR⁴—C₁₋₄alkyl-O—SO₂CH₃,4-CH₃-Ph-SO₂—O—C₁₋₄alkyl-NR⁴—, and —NR⁴—C₁₋₄alkyl-OH, wherein R⁴ is H ormethyl;or a pharmaceutically acceptable salt or a solvate thereof.

In compounds of Formula (P-1), when either of R¹ or R³ are —[N(CH₃)₃]⁺,suitable anionic counterions include, but are not limited totrifluoroacetate (—[OC(O)CF₃]⁻), an organic sulfonate (e.g.C₁₋₄alkylsulfonate, or phenylsulfonate wherein the phenyl may beoptionally substituted with a C₁₋₄alkyl, halo, or a nitro group) andtartrate. Particular examples of C₁₋₄alkylsulfonate includemethanesulfonate (mesylate), 4-methylbenzenesulfonate (tosylate),4-bromobenzensulfonate and 4-nitrobenzenesulfonate. In particular theanionic counterion is selected from trifluoroacetate, tosylate, andmesylate.

The invention also relates to the reference materials of compounds ofFormula (I) or (I′), corresponding to the correspondingnon-radiolabelled compounds, herein referred to as compounds of Formula[¹⁹F]-(I)

whereinR² is methyl, and either R¹ is F and R³ is H, or R¹ is H and R³ is F; orR¹ and R³ are both H, and R² is selected from the group consisting of—C₁₋₄alkyl-F, —OC₁₋₄alkyl-F, and —NR⁴—C₁₋₄alkyl-F, wherein R⁴ is H ormethyl;or a pharmaceutically acceptable salt or a solvate thereof.

The invention also relates to a pharmaceutical composition comprising acompound of Formula (I), in particular a compound of Formula (I′), or apharmaceutically acceptable salt thereof and a pharmaceuticallyacceptable carrier or diluent. In particular, said pharmaceuticalcomposition is a diagnostic pharmaceutical composition. Saidpharmaceutical composition is in particular, a sterile solution. Thus,illustrative of the invention is a sterile solution comprising acompound of Formula (I), in particular a compound of Formula (I′), asdescribed herein.

The invention further relates to the use of a compound of Formula (I),in particular a compound of Formula (I′), as an imaging agent.Exemplifying the invention is a use of a compound of Formula (I), inparticular a compound of Formula (I′), as described herein, for, or amethod of, imaging a tissue or a subject, in vitro or in vivo.

In particular, the invention relates to a compound of Formula (I), inparticular a compound of Formula (I′), for use in binding and imagingtau aggregates in patients suffering from, or suspected to be sufferingfrom, a tauopathy. Particular tauopathies are, for example, Alzheimer'sdisease, tangle-only dementia (TD), argyrophilic grain disease (AGD),progressive supranuclear palsy (PSP), corticobasal degeneration (CBD),Pick disease (PiD), and frontotemporal dementia and parkinsonism linkedto chromosome 17 (FTDP-17). In particular, the tauopathy is Alzheimer'sdisease.

The invention further relates to a compound of Formula (I), inparticular a compound of Formula (I′), for diagnostic imaging of tauaggregates in the brain of a subject, and to the use of the compound ofFormula (I), in particular the compound of Formula (I′), in binding andimaging tau aggregates in patients suffering from, or suspected to besuffering from, a tauopathy. Particular tauopathies are, for example,Alzheimer's disease, tangle-only dementia (TD), argyrophilic graindisease (AGD), progressive supranuclear palsy (PSP), corticobasaldegeneration (CBD), Pick disease (PiD), and frontotemporal dementia andparkinsonism linked to chromosome 17 (FTDP-17). In particular, thetauopathy is Alzheimer's disease.

The invention also relates to a method for imaging a tissue or asubject, comprising contacting with or providing or administering adetectable amount of a labelled compound of Formula (I), in particular alabelled compound of Formula (I′), as described herein to a tissue, or asubject, and detecting the compound of Formula (I), in particular thecompound of Formula (I′).

Further exemplifying the invention is a method of imaging a tissue, or asubject, comprising contacting with or providing to a tissue, or asubject, a compound of Formula (I), in particular a compound of Formula(I′), as described herein, and imaging the tissue, or subject with apositron-emission tomography imaging system.

Additionally, the invention refers to a process for the preparation of acompound of Formula (I′-a) or (I′-b), referred to in particularhereinbelow as (I′-a1), (I′-a2), (I′-b1), (I′-b2), (I′-b3), or apharmaceutically acceptable salt or a solvate thereof as describedherein, comprising

(a) the step of reacting a compound of Formula (P-a1) or apharmaceutically acceptable salt or a solvate thereof, as definedherein, with a source of fluoride ¹⁸F⁻ under suitable conditions, or

(b) the step of reacting a compound of Formula (P-a2) or apharmaceutically acceptable salt or a solvate thereof, as definedherein, with a source of fluoride ¹⁸F⁻ under suitable conditions, or

(c) the step of reacting a compound of Formula (P-b 1) or apharmaceutically acceptable salt thereof, as defined herein, with anactivating reagent such as methanesulfonyl chloride or 4-toluenesulfonylchloride in the presence of a base such as triethylamine orN,N-diisopropylethylamine (DIPEA), and subsequently reacting theresulting methanesulfonate or 4-toluenesulfonate with a source offluoride ¹⁸F⁻ under suitable conditions, or(d) the step of reacting a compound of Formula (P-b2) or apharmaceutically acceptable salt thereof, as defined herein, with anactivating reagent such as methanesulfonyl chloride or 4-toluenesulfonylchloride in the presence of a base such as triethylamine or DIPEA, andsubsequently reacting the resulting methanesulfonate or4-toluenesulfonate with a source of fluoride ¹⁸F⁻ under suitableconditions, or(e) the step of reacting a compound of Formula (P-b3) or apharmaceutically acceptable salt thereof, as defined herein, with anactivating reagent such as methanesulfonyl chloride or 4-toluenesulfonylchloride in the presence of a base such as triethylamine or DIPEA, andsubsequently reacting the resulting methanesulfonate or4-toluenesulfonate with a source of fluoride ¹⁸F⁻ under suitableconditions.

Typical conditions for the activation of the precursors of Formulae(P-b1), (P-b2) and (P-b3), include for instance, methanesulfonylchloride as activating agent, dry dimethyl sulfoxide (DMSO) as solvent,at room temperature for a sufficient period of time to drive thereaction to completion, typically, 10 minutes. The skilled person willunderstand that when R⁴ is H in (P-b3), the preparation of the compoundof Formula (I′-b3) will include the additional steps of protecting theamine functionality with a suitable protecting group, such as forexample tert-butyloxycarbonyl (Boc) or alternative suitable amineprotecting group, and subsequently cleaving such protecting group, usingtypically trifluoroacetic acid (TFA) when the protecting group is Boc.

A suitable source of ¹⁸F⁻ is, for example4,7,13,16,21,24-hexaoxa-1,10-diazabicyclo[8.8.8]hexacosane potassiumfluoride-[¹⁸F] (1:1) (also referred to as [¹⁸F]KF.K222). Suitableconditions include, those appropriate for nucleophilic substitutionknown in the art, for example, using DMSO or DMF as solvent, inparticular DMSO, under conventional heating or microwave irradiation(e.g. 50 W), for example at about 90-160° C., or at about 120-160° C.,in particular at about 90 or 160° C., for a sufficient period of time toenable the reaction to proceed to completion, for example 10 min whenthe reaction is performed under microwave irradiation.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows in vitro autoradiography (ARG) using [¹⁸F]Co. No. 1 (7.4kBq/500 μL/slice) on a slice of the visual cortex of a patient with AD(Braak stage VI) (A). Adjacent slices were immunostained for taupathology (B, AT8) and for amyloid beta pathology (C, 4G8).

FIG. 2 shows a comparison between the ARG shown in FIG. 1 (A), andadjacent human AD brain slices incubated with [¹⁸F]Co. No. 1 (7.4kBq/500 μL/slice) in the presence of authentic reference compound Co.No. 1 (B) or T808 (C) at 1 μmol/L.

FIG. 3 shows incubation with 10 nM [³H]Co. No. 1 on human basal ganglia(striatal) tissue (A), followed by treatment with 10 μM Co. No. 1 (B) or10 μM THK5351 (C). FIG. 3D shows incubation with 10 nM [³H]Co. No. 1 onhuman AD tissue (parietal-temporal cortex, Braak stage VI) havingconfirmed tau and amyloid pathology by IHC as shown in FIG. 4. FIG. 3Eshows incubation with 10 nM [³H]Co. No. 1 on human AD tissue(lateral-occipital gyrus, Braak stage 0), with confirmed amyloidpathology but no tau pathology by IHC as shown in FIG. 5. FIG. 3F showsincubation with 10 nM [³H]THK5351 on human basal ganglia tissue, andtreatment with 10 μM Co. No. 1 (G) or 10 μM THK5351 (H). FIG. 3I showsincubation with [³H]THK-5351 on human AD tissue (parietal-temporalcortex, Braak stage VI) with confirmed tau and amyloid pathology asshown in FIG. 4. FIG. 3J shows incubation with 10 nM [³H]THK5351 onhuman AD tissue (lateral-occipital gyrus, Braak stage 0) with confirmedamyloid pathology but no tau pathology as shown in FIG. 5. FIG. 3K showsincubation with 3 nM [³H]-AV-45 on human basal ganglia (K), theaforementioned human AD tissue containing both amyloid beta and taupathology (L) and the aforementioned human AD tissue containing amyloidbeta but no taupathology (M).

FIG. 4 shows 4G8 and AT8 IHC of amyloid and tau pathology in AD brainslices from parietal-temporal cortex region of the same patient as theslices used for the images in FIG. 3.

FIG. 5 shows an adjacent human AD brain slice (lateral-occipital gyrus,female) to the one used in FIG. 3E and FIG. 3J. The presence ofβ-amyloid plaques (A, B) and absence of tau tangles (C, D) was confirmedwith immunohistochemistry using respectively 4G8 and AT8 antibodies.

FIG. 6 shows IHC confirmation of the expression of MAO-B (left) andMAO-A (right) in the human basal ganglia tissue used for in vitrobinding shown in FIG. 3.

FIG. 7 shows incubation with 0.2 mCi/mL [¹⁸F]Co. No. 1 on human ADtissue slices (parieto-temporal cortex, Braak stage VI) (A), andtreatment with 10 μM Clorgiline (B), 10 μM Deprenyl (C) and 10 μM Co.No. 1 (D).

FIG. 8 shows AT8 and 4G8 IHC of respectively tau and amyloid pathologyin adjacent AD brain slices (parietal-temporal cortex, Braak stage VI)to the ones referred to in FIG. 7.

FIG. 9 shows μPET time-activity curves expressed as standardized uptakevalues (SUV) for [¹⁸F]Co. No. 1 and FIG. 10 shows μPET time-activitycurves for [¹⁸F]T807 (C) in the whole brain of three female Wistar rats.Three separate studies are shown: a baseline scan (only tracer), apre-treatment experiment (cold Co. No. 1 or T807, 10 mg/kg injectedsubcutaneously 60 min prior to radiotracer injection), and a chase study(cold Co. No. 1 or T807, 1 mg/kg injected intravenously 30 min afterradiotracer injection).

FIG. 11 shows % SUV_(max) curves of small animal PET time-activitycurves of [¹⁸F]Co. No. 1 and [¹⁸F]T807 in the total brain of a Wistarrat.

FIG. 12 shows μPET time-activity curves for [¹⁸F]Co. No. 1 and FIG. 13shows μPET time-activity curves for [¹⁸F]T807 in the whole brain, corpuscallosum, cerebellum, enthorinal cortex and skull of a rhesus monkey.

FIG. 14 shows % SUV_(max) curves of small animal μPET time-activitycurves of [¹⁸F]Co. No. 1 and [¹⁸F]T807 in the total brain of a rhesusmonkey.

DETAILED DESCRIPTION OF THE INVENTION

In an embodiment, the compound of Formula (I) is in particular acompound of Formula (I-a)

whereinat least one atom is radioactive, and wherein R¹ is F and R³ is H, or R¹is H and R³ is F;or a pharmaceutically acceptable salt or a solvate thereof.

More in particular, the compound of Formula (I) is a compound of Formula(I′-a)

whereinR¹ is ¹⁸F and R³ is H, or R¹ is H and R³ is ¹⁸F;or a pharmaceutically acceptable salt or a solvate thereof.

In another embodiment, the compound of Formula (I) is in particular acompound of Formula (I-b)

whereinat least one atom is radioactive, and wherein R² is selected from thegroup consisting of —C₁₋₄alkyl-F, —OC₁₋₄alkyl-F, and —NR⁴—C₁₋₄alkyl-F,wherein R⁴ is H or methyl;or a pharmaceutically acceptable salt or a solvate thereof.

More in particular, the compound of Formula (I) is a compound of Formula(I′-b)

whereinR² is selected from the group consisting of —C₁₋₄alkyl-¹⁸F,—OC₁₋₄alkyl-¹⁸F, and —NR⁴—C₁₋₄alkyl-¹⁸F, wherein R⁴ is H or methyl;or a pharmaceutically acceptable salt or a solvate thereof.

In an embodiment, the compound of Formula (I), in particular of Formula(I′), is

or a pharmaceutically acceptable salt or a solvate thereof.

In another particular embodiment, the precursor compound for thesynthesis of the compound of Formula (I) or (I′), as previously defined,is in particular a compound of Formula (P-1)

whereinR² is methyl, and either R¹ is selected from the group consisting of Br,—NO₂, —[N(CH₃)₃]⁺, and 4-Me-Ph-SO₂—O—, and R³ is H, or R³ is H and R³ isselected from the group consisting of Br, —NO₂, —[N(CH₃)₃]⁺, and4-Me-Ph-SO₂—O—; orR¹ and R³ are both H, and R² is selected from the group consisting of—C₁₋₄alkyl-OH, —OC₁₋₄alkyl-OH, and —NR⁴—C₁₋₄alkyl-OH, wherein R⁴ is H ormethyl;or a pharmaceutically acceptable salt or a solvate thereof.

The invention also relates to the reference material corresponding tothe non-radiolabelled compound 1, corresponding to the [¹⁹F]-compound

or a pharmaceutically acceptable salt or a solvate thereof.

In another particular embodiment, the compound of Formula [¹⁹F]-(I) isin particular a compound of Formula [¹⁹F]-(I-a)

whereinR¹ is F and R³ is H, or R¹ is H and R³ is F;or a pharmaceutically acceptable salt or a solvate thereof.

In another particular embodiment, the compound of Formula [¹⁹F]-(I) isin particular a compound of Formula (I-b)

whereinR² is selected from the group consisting of —C₁₋₄alkyl-F, —OC₁₋₄alkyl-F,and —NR⁴—C₁₋₄alkyl-F, wherein R⁴ is H or methyl;or a pharmaceutically acceptable salt or a solvate thereof.

[¹⁹F]-Co. No. 1 has shown potent binding (pIC₅₀ 8.24) to extracted humantau aggregates in a radiolabel displacement assay using an internallymade tritium analogue of compound T-808 (T-808 is developed by Siemens,see for example, J. Alzheimers Dis. 2014, 38, 171-184), and which isreferred to herein as [³H]-T808. In addition, Co. No. 1 shows weakbinding to extracted human amyloid-beta aggregates (pIC₅₀ 5.18) in aradiolabel displacement assay using an internally made tritium analogueof Florbetapir (also known as Amyvid® from Eli Lilly and Co., or AV-45,see for example, J. Nucl. Med. 2010, 51, 913-920), and which is referredto herein as [³H]-AV-45. A description of the protocols is providedhereinafter.

[³H]-T808 was obtained by subjecting a solution of the bromo precursor(1 eq.) in methanol to catalytic tritiation over palladium on carbon(5%) in the presence of diisopropylethylamine (5 eq.) at roomtemperature. The bromo precursor was obtained by bromination of T808with N-bromosuccinimide (1 eq.) in acetonitrile.

[³H]-AV-45 was obtained by Iridium catalyzed (Crabtree's catalyst)tritium exchange of AV-45 dissolved in dichloromethane.

As already mentioned, the compound of Formula (I), in particular thecompound of Formula (I′), and compositions comprising the compound ofFormula (I), in particular the compound of Formula (I′), can be used forimaging a tissue, or a subject, in vitro or in vivo. In particular, theinvention relates to a method of imaging or quantifying tau aggregatesin a tissue, or a subject in vitro or in vivo.

In particular, the method of imaging tau aggregates comprises providinga subject, in particular a patient, with a detectable quantity of acompound of Formula (I), in particular a compound of Formula (I′).

Further, the invention relates to a method of imaging tau-aggregatedeposits comprising the steps of providing a subject with a detectablequantity of a compound of Formula (I), in particular a compound ofFormula (I′), allowing sufficient time for the compound of Formula (I),in particular the compound of Formula (I′), to be associated with tauaggregate deposits, and detecting the compound associated with tauaggregate deposits.

When the method is performed in vivo, the compound of Formula (I), inparticular the compound of Formula (I′), can be administeredintravenously, for example, by injection with a syringe or by means of aperipheral intravenous line, such as a short catheter. The compound ofFormula (I), in particular the compound of Formula (I′), or a sterilesolution comprising a compound of Formula (I), in particular a compoundof Formula (I′), may in particular be administered by intravenousadministration in the arm, into any identifiable vein, in particular inthe back of the hand, or in the median cubital vein at the elbow.

Thus, in a particular embodiment, the invention relates to a method ofimaging a subject, comprising the intravenous administration of acompound of Formula (I), in particular a compound of Formula (I′), asdefined herein, or a composition, in particular, a sterile formulation,comprising a compound of Formula (I), in particular a compound ofFormula (I′), to the subject, and imaging the subject with apositron-emission tomography imaging system.

In a further embodiment, the invention relates to a method ofquantifying tau aggregation deposits in a subject, comprising theintravenous administration of a compound of Formula (I), in particular acompound of Formula (I′), or a composition comprising a compound ofFormula (I), in particular a compound of Formula (I′), to the subject,and imaging with a positron-emission tomography imaging system.

The compound is provided to a subject in a detectable quantity and aftersufficient time has passed for the compound to become associated withthe tau aggregation deposits, the labelled compound is detectednoninvasively.

Definitions

As used herein, the term “composition” is intended to encompass aproduct comprising the specified ingredients in the specified amounts,as well as any product which results, directly or indirectly, fromcombinations of the specified ingredients in the specified amounts. Theterm “C₁₋₄alkyl” shall denote a straight or branched saturated alkylgroup having 1, 2, 3 or 4 carbon atoms, respectively e.g. methyl, ethyl,1-propyl, 2-propyl, butyl and the like.

Addition salts of the compounds according to the invention also intendedto be encompassed within the scope of this invention.

Acceptable salts of the compounds of the invention are those wherein thecounterion is pharmaceutically acceptable. However, salts of acids andbases which are non-pharmaceutically acceptable may also find use, forexample, in the preparation or purification of a pharmaceuticallyacceptable compound. All salts, whether pharmaceutically acceptable ornot, are included within the ambit of the present invention. Thepharmaceutically acceptable salts are defined to comprise thetherapeutically active non-toxic acid addition salt forms that thecompounds according to the invention are able to form. Said salts can beobtained by treating the base form of the compounds according to theinvention with appropriate acids, for example inorganic acids, forexample hydrohalic acid, in particular hydrochloric acid, hydrobromicacid, sulphuric acid, nitric acid and phosphoric acid; organic acids,for example acetic acid, hydroxyacetic acid, propanoic acid, lacticacid, pyruvic acid, oxalic acid, malonic acid, succinic acid, maleicacid, fumaric acid, malic acid, tartaric acid, citric acid,methanesulfonic acid, ethanesulfonic acid, benzensulfonic acid,p-toluenesulfonic acid, cyclamic acid, salicylic acid, p-aminosalicylicacid and pamoic acid.

Conversely, said salt forms can be converted into the free base form bytreatment with an appropriate base.

In addition, some of the compounds of the present invention may formsolvates with water (i.e., hydrates) or common organic solvents, andsuch solvates are also intended to be encompassed within the scope ofthis invention.

The term “subject” as used herein, refers to a human, who is or has beenthe object of treatment, observation or experiment. Unless otherwisestated, “subject” includes non-symptomatic humans, presymptomatic humansand human patients.

Preparation

The compounds according to the invention can generally be prepared by asuccession of steps, each of which is known to the skilled person. Inparticular, the compounds can be prepared according to the followingsynthesis methods.

Compounds of Formula [¹⁹F]-(I-a) or [¹⁹F]-(I-b) as disclosed herein canbe prepared by a reaction of 6-bromo-isoquinoline with an appropriate2-amino-pyridine compound of Formula (II-a) wherein all variables are asdescribed herein for [¹⁹F]-(I)

under Buchwald-Hartwig amination conditions, wherein all variables areas described herein for [¹⁹F]-(I), or alternatively, by a reaction of acompound of Formula (II-a′) wherein R¹ is —NO₂ and R³ is H, or R¹ is Hand R³ is —NO₂, with a fluoride source, such as KF, in a reaction inertsolvent, such as DMSO, under thermal conditions,

or alternatively, by a reaction of isoquinolin-6-amine with anappropriate 2-chloro-pyridine compound of Formula (II-b) wherein allvariables are as described herein for [¹⁹F]-(I)

under Buchwald-Hartwig amination conditions.

The compound of Formula (II-a′) can be prepared for example, by reactionof isoquinolin-6-amine with 6-bromo-3-methyl-2-nitropyridine or2-bromo-5-methyl-4-nitropyridine under Buchwald-Hartwig aminationconditions.

Applications

The compounds according to the present invention find variousapplications for imaging tissues, or a subject, both in vitro and invivo. Thus, for instance, they can be used to map the differentialdistribution of tau aggregate deposits in subjects of different age andsex. Further, they allow one to explore for differential distribution oftau aggregate deposits in subjects afflicted by different diseases ordisorders, including Alzheimer's disease, but also other diseases causedby tau aggregate deposits, i.e. other tauopathies.

Thus, excess distribution may be helpful in diagnosis, case finding,stratification of subject populations, and in monitoring diseaseprogression in individual subjects, particularly when anti-tautreatments, e.g. antibodies, become available. Since the radioligand isadministered in trace amounts, i.e. in detectable amounts for PETimaging, no therapeutic effect may be attributed to the administrationof the radioligands according to the invention.

Experimental Part

Chemistry

As used herein, the term “ACN or MeCN” means acetonitrile, “aq.” meansaqueous, “tBuOH” means tert-butanol, “DCM” means dichloromethane, “DIPE”means diisopropyl ether, “DIPEA” means N,N-diisopropylethylamine, “DMF”means N,N-dimethylformamide, “DMSO” means dimethyl sulfoxide, “Et₂O”means diethyl ether, “EtOAc” means ethyl acetate, “h” means hours,“HPLC” means high-performance liquid chromatography, “LCMS” means liquidchromatography/mass spectrometry, “MeOH” means methanol, “min” meansminutes, “m.p.” means melting point, “org” means organic, “Pd₂(dba)₃”means tris(dibenzylideneacetone)dipalladium(0), “prep” meanspreparative, “rm/RM” means reaction mixture, “rt/RT” means roomtemperature”, “R_(t)” means retention time (in minutes), “sat.” meanssaturated, “sol.” means solution, “TFA” means trifluoroacetic acid,“THF” means tetrahydrofuran, “XantPhos” means4,5-bis(diphenyl-phosphino)-9,9-dimethylxanthene.

Thin layer chromatography (TLC) was carried out on silica gel 60 F254plates (Merck) using reagent grade solvents. Open column chromatographywas performed on silica gel, mesh 230-400 particle size and 60 Å poresize (Merck) under standard techniques. Automated flash columnchromatography was performed using ready-to-connect disposablecartridges purchased from Grace (GraceResolv™ cartridges) or TeledyneISCO (RediSep® (cartridges), on irregular silica gel, particle size35-70 μm on an ISCO CombiFlash or Biotage Isolera™ Spektra apparatus.

Nuclear Magnetic Resonance (NMR): For a number of compounds, ¹H NMRspectra were recorded either on a Bruker Ultrashield AV300, Bruker DPX360 MHz NMR or Bruker Avance III 400 MHz NMR spectrometer with standardpulse sequences, operating at 300 MHz, 360 MHz and 400 MHz,respectively. Samples were dissolved in DMSO-d₆ or CDCl₃ and transferredin 5 mm NMR tubes for the measurement. Chemical shifts (δ) are reportedin parts per million (ppm) downfield from tetramethylsilane (TMS), whichwas used as internal standard.

HPLC purifications were carried out on a GILSON Semi-Preparative System,operated by Trilution software, equipped with a Phenomenex Gemini C18100A column (100 mm long×30 mm I.D.; 5 μm particles) at RT ° C., with aflow rate of 40 mL/min using a gradient elution in 20 min as indicatedin the synthetic protocols. The injection volume was 8000 μL.Acquisition frequency was set to 284 nm for the UV-Dual detector.

Several methods for preparing the compounds of this invention areillustrated in the following examples, which are intended to illustratebut not to limit the scope of the present invention. Unless otherwisenoted, all starting materials were obtained from commercial suppliersand used without further purification.

A. Synthesis of Intermediates

Preparation of Intermediate 1

Tetramethyltin (0.736 mL, 5.309 mmol) was added to a mixture of5-bromo-4-fluoropyridin-2-amine (0.338 g, 1.77 mmol, CAS 944401-69-8),bis(triphenylphosphine)palladium(II) chloride (0.062 g, 0.0885 mmol) andLiCl (0.300 g, 7.078 mmol) in 10 mL DMF. This mixture was sealed in atube after degassing with nitrogen. Next, the mixture was stirred at120° C. for 18 hours, after which it was diluted with water. Next, asaturated aqueous solution of KF was added and the aq. layer wasextracted with EtOAc. The combined org layers were dried (MgSO₄),filtered and the solvents evaporated in vacuo. The crude was purified byflash column chromatography (silica; DCM/MeOH, 100/0 to 95/5). Thedesired fractions were collected and concentrated in vacuo providingintermediate 1 (0.190 g, 79%).

Preparation of Intermediate 2

Method 1: To a stirred solution of 6-aminoisoquinoline (18.3 g, 127.1mmol) in toluene (400 mL) were added 2-bromo-5-methyl-4-nitropyridine(23.0 g, 105.9 mmol), Xantphos (2.45 g, 4.24 mmol), Pd₂(dba)₃ (1.94 g,2.12 mmol), tBuOK (16.6 g, 148.3 mmol) under nitrogen. Nitrogen wasbubbled through the mixture for 5 min, and then the vial was sealed andheated at 100° C. for 2 h. The mixture was filtered, and the filter cakewashed with EtOAc until all product was extracted. Water was added tothe filtrate and the org layer was separated, dried (MgSO₄), filteredand concentrated. The residue was taken up in water, triturated for 1 h,filtered, and dried in vacuo at 55° C. The resulting brown solid(dissolved in MeOH at pH 4) was purified via Prep HPLC (Stationaryphase: RP XBridge Prep C18 OBD-10 μm, 50×150 mm, Mobile phase: 0.5%NH₄OAc solution in water+10% CH₃CN, MeOH). The pure fractions werecombined and the organic component of the eluent was evaporated. A redprecipitate formed which was filtered, washed with water and dried. Theresulting precipitate was stirred in DCM/MeOH/1 N NaOH (2 L/0.2 L/1 L)until everything dissolved, and the org layer was separated. The aq.layer was extracted once more with DCM. The combined org layers weredried (MgSO₄), filtered and evaporated. The residue was triturated in abiphasic mixture of ether and water, filtered and dried in vacuo for 2days at 50° C. and 4 h in a lyophilizer at rt to give intermediate 2 asorange crystals (6.11 g, 18%). ¹H NMR (400 MHz, DMSO-d₆) δ ppm 2.40 (s,3H) 7.50 (s, 1H) 7.62-7.71 (m, 2H) 8.01 (d, J=9.0 Hz, 1H) 8.36 (d, J=5.7Hz, 1H) 8.45 (s, 2H) 9.09 (s, 1H) 9.97 (s, 1H)

Method 2: To a stirred solution of 6-aminoisoquinoline (7.62 g, 52.85mmol) in 200 mL toluene were added 2-bromo-5-methyl-4-nitropyridine(11.47 g, 52.85 mmol), Xantphos (0.611 g, 1.057 mmol), Pd₂(dba)₃ (0.484g, 0.529 mmol) and tBuOK (8.30 g, 74.00 mmol). Nitrogen was bubbledthrough the mixture for 5 min, and then the vial was sealed and heatedat 100° C. for 2 h, and again for 3 h at 110° C. EtOAc was added to themixture after which it was stirred for 30 min. Next the mixture wasfiltered over dicalite, and the filter cake washed with EtOAc until allproduct was extracted. Water was added to the filtrate and the org layerwas separated. The aq. layer was extracted with EtOAc until no productremained (LCMS control). The combined org layers were washed with brine,dried (MgSO₄), filtered and concentrated. The residue was taken up inwater/DIPE, triturated overnight, filtered and dried in vacuo at 55° C.yielding intermediate 2 (10.5 g, 71%, 57% pure), which was used as suchfor the next reaction step.

Preparation of Intermediate 3

Trimethylboroxine (0.256 mL, 1.83 mmol) was added to a stirred solutionof 5-bromo-6-fluoropyridin-2-amine (0.291 g, 1.52 mmol, CAS944401-65-4), tetrakis(triphenylphosphine)palladium(0) (0.176 g, 0.152mmol) and Cs₂CO₃ (0.993 g, 3.047 mmol) in 1,4-dioxane (5.3 mL) whilebubbling nitrogen. The reaction was stirred at 105° C. for 16 h undernitrogen. Water and EtOAc were added. The phases were separated andorganic was dried over MgSO₄, filtered and evaporated. The crude productwas purified by flash column chromatography (silica; EtOAc in Heptane,0/100 to 50/50). The desired fractions were collected and concentratedin vacuo to yield intermediate 3 as a yellow solid (0.151 g, 79%).

Preparation of Intermediate 4

Deoxo-Fluor® (50% in toluene, 1.67 mL, 3.807 mmol) was added dropwise toa solution of 2-chloro-5-(2-fluoroethyl)pyridine (0.400 g, 2.538 mmol,CAS 117528-28-6) in dry DCM (15 mL) at 0° C. After 1 min, the cold bathwas removed and reaction mixture was stirred at room temperature for 2h. The mixture was diluted with aq. sat. NaHCO₃ and extracted with DCM.The organic layer was washed with water, dried with (MgSO₄), filteredand the solvents evaporated in vacuo. The crude product was purified byflash column chromatography (silica; EtOAc in heptane from 0/100 to70/30). The desired fractions were collected and concentrated in vacuoyielding intermediate 4 (0.193 g, 45%).

Preparation of Intermediate 5

NaOH (0.064 g, 1.599 mmol) was added to a mixture of[(6-chloropyridin-3-yl)oxy]acetic acid (0.300 g, 1.599 mmol, CAS234109-28-5) in a mixture of CH₃CN (4 mL) and water (4 mL). The mixturewas heated at 85° C. for 1 h. The solvent was concentrated in vacuoproviding intermediate 5 (335 mg, 95%) as a white solid, which was usedas such in the next step.

Preparation of Intermediate 6

Selectfluor® (1.116 g, 3.15 mmol) was added to a solution ofintermediate 5 (0.314 g, 1.5 mmol) in a mixture of water (7.5 mL) andCH₃CN (7.5 mL) previously degassed.Tris(2,2′-bipyridyl)dichlororuthenium(II)hexahydrate (0.056 g, 0.075mmol) was added. The reaction was irradiated by a 500 W visible lightshipyard lamp, placed at 30 cm from the reaction, for 1 h. After turningoff the light, water and diethylether were poured on the reaction media.The two phases were separated and the aqueous one was extracted withEt₂O. The combined organic phases were dried over MgSO₄, filtered, andthe solvent was evaporated under reduced pressure. The crude wasfiltered over silica gel with Et₂O as eluent and concentrated in vacuoto yield intermediate 6 as a beige oily solid (0.167 g, 54%).

Preparation of Intermediate 7

A stirred solution of 5-amino-2-chloropyridine (1.00 g, 7.778 mmol) inTHF (27 mL) was cooled to 0° C., then 2-nitrobenzenesulfonylchloride(1.72 g, 7.778 mmol) and pyridine (0.942 mL, 11.668 mmol) were added tothe solution. The resulting mixture was allowed to warm to rt andstirred for 3 hours. 2-Nitrobenzenesulfonylchloride (0.52 g, 2.334 mmol)and pyridine (0.314 mL, 3.889 mmol) were added at 0° C. and the mixturewas stirred at rt overnight. Water was then added to the mixture, andthe aq. layer was extracted with EtOAc. The extract was washed withsaturated aq. NaHCO₃ and brine. The org layer was dried over MgSO₄,filtered, and evaporated. The residue was purified by flash columnchromatography (silica; EtOAc in Heptane, 0/100 to 10/90). The desiredfractions were collected and concentrated in vacuo to yield intermediate7 (2.22 g, 91%).

Preparation of Intermediate 8

Intermediate 7 (1.1 g, 3.506 mmol) and 2-fluoroethyl4-methylbenzenesulfonate (0.918 g, 4.208 mmol, CAS 383-50-6) weredissolved in DMF (10.6 mL). Cs₂CO₃ (1.942 g, 5.961 mmol) was added undernitrogen atmosphere. The mixture was heated to 85° C. overnight. Aftercooling to ambient temperature, the dark brown suspension was dilutedwith water and extracted with EtOAc. The org layer was washed withbrine, dried over MgSO₄, filtered and concentrated. The product waspurified by flash column chromatography (silica; EtOAc in heptane, 0/100to 20/80). The desired fractions were collected and concentrated invacuo providing intermediate 8 (0.687 g, 54%).

Preparation of Intermediate 9

Intermediate 7 (0.700 g 2.231 mmol) and 3-fluoropropyl4-methylbenzenesulfonate (0.674 g, 2.901 mmol, CAS 312-68-5) weredissolved in DMF (6.7 mL). Cs₂CO₃ (1.45 g, 4.463 mmol) was added and themixture was heated to 85° C. overnight under nitrogen atmosphere.3-Fluoropropyl 4-methylbenzenesulfonate (0.363 g, 1.562 mmol) and Cs₂CO₃(0.727 g, 2.231 mmol) were added and the mixture was heated at 85° C.overnight. After cooling to ambient temperature, the dark brownsuspension was diluted with water and extracted with EtOAc. The organiclayer was washed with brine, dried over MgSO₄, filtered andconcentrated. The product was purified by flash column chromatography(silica; EtOAc in heptane, 0/100 to 15/85). The desired fractions werecollected and concentrated in vacuo yielding intermediate 9 (0.395 g,42%).

Preparation of Intermediate 10

To a stirred solution of intermediate 8 (0.687 g 1.91 mmol) in 11.6 mLDMF, LiOH.H₂O (0.491 g, 11.46 mmol) and 2-mercaptoethanol (0.164 mL,2.33 mmol) were added at 0° C. The reaction mixture was stirred at rtfor 2 hours. Water was added and the mixture was extracted with EtOAc.The extract was washed with water and dried over MgSO₄. Concentrationunder reduced pressure gave oily residues. The crude was purified byflash chromatography (silica gel, EtOAc in Heptane, from 0/100 to30/70).

The desired fractions were collected and concentrated in vacuo to yieldintermediate 10 as a yellow oil (0.276 g, 83%).

Preparation of Intermediate 11

Intermediate 10 (0.200 g, 1.145 mmol) was added to a stirred solution ofNaH (60% dispersion in mineral oil, 0.069 g, 1.718 mmol) in DMF (9.2 mL)at 0° C. under a nitrogen atmosphere. After being stirred for 30 min atthis temperature, CH₃I (0.134 mL, 1.833 mmol) was added and stirring wascontinued overnight at rt. NaH (60% dispersion in mineral oil, 0.032 g,0.802 mmol) was added at 0° C. and the mixture was stirred 30 min. CH₃I(0.032 mL, 0.573 mmol) was added and the mixture was stirred at rt for 2h. The mixture was diluted with water and extracted with EtOAc. The orglayer was dried (MgSO₄), filtered and evaporated in vacuo. The crudeproduct was purified by flash column chromatography (EtOAc in heptane0/100 to 15/85). The desired fractions were collected and concentratedin vacuo to yield intermediate 11 (0.137 g, 63%).

Preparation of Intermediate 12

NaH (60% dispersion in mineral oil, 0.178 g, 4.46 mmol) was added to astirred solution of tert-butyl (6-chloropyridin-3-yl)carbamate (1.36 g,75% pure, 4.46 mmol, CAS 171178-45-3) in dry DMF (7 mL) at 0° C. underN₂. The mixture was stirred at 0° C. for 15 min. Then1-bromo-3-fluoropropane (0.491 mL, 5.353 mmol) was added at 0° C. andthe mixture was allowed to warm to rt and stirred overnight. More1-bromo-3-fluoropropane (0.327 mL, 3.568 mmol) and NaH (60% dispersionin mineral oil, 0.178 g, 4.46 mmol) were added and the mixture wasstirred at rt for 4 h. Additional 1-bromo-3-fluoropropane (0.818 mL,8.921 mmol) and NaH (60% dispersion in mineral oil, 0.178 g, 4.46 mmol)were added and the mixture was stirred at rt overnight. The reactionmixture was taken up with DCM and washed with a saturated solution ofNH₄Cl. The organic layer was separated, dried over MgSO₄, filtered andconcentrated in vacuo. The residue was purified by flash columnchromatography (silica; EtOAc in heptane 40% to 100%). The desiredfractions were recovered and the solvents were evaporated in vacuo toyield intermediate 12 as a colorless oil (1.71 g, 75% pure, quantitativeyield).

Preparation of Intermediate 13

Method 1: TFA (4 mL, 53.3 mmol) was added to a mixture of intermediate12 (1.71 g, 5.33 mmol, 75% pure) in 20 mL DCM at 0° C. and then themixture was stirred at rt for 150 min. Next, the mixture was dilutedwith DCM and washed with an aq. sat. sol. of NaHCO₃. The org layer wasseparated, dried (MgSO₄), filtered and concentrated in vacuo to provideintermediate 13 (1.91 g, 53% pure, quantitative), which was used assuch.

Method 2: To a stirred solution of intermediate 7 (0.395 g, 0.93 mmol)in DMF (5.6 mL), LiOH.H₂O (0.239 g, 5.58 mmol) and 2-mercaptoethanol(0.798 mL, 1.134 mmol) were added at 0° C. The reaction mixture wasstirred at rt for 2 h, after which it was extracted with EtOAc. Thecombined org layers were washed with water, dried over MgSO₄ andfiltered. Concentration under reduced pressure gave an oily residuewhich was purified by flash chromatography (silica gel, EtOAc inheptane, from 0/100 to 30/70). The desired fractions were collected andconcentrated in vacuo to yield intermediate 13 as a yellow oil (0.168 g,96%).

Preparation of Intermediate 14

Method 1: Formaldehyde (37% in water, 2.185 mL, 29.162 mmol) was addedto a solution of intermediate 13 (obtained from method 1, 1.91 g, 9.721mmol, 53% pure) in a mixture of acetic acid (0.835 mL) and MeOH (20 mL).Then NaBH₃CN (1.83 g, 29.162 mmol) was added portion wise. The mixturewas stirred at rt overnight (15 h). A sat. sol. of NaHCO₃ was added andthe mixture was extracted with EtOAc. The org layers were dried overMgSO₄, filtered and concentrated. The crude product was purified byflash column chromatography (silica; EtOAc in heptane, 0/100 to 80/20).The desired fractions were collected and concentrated in vacuo to yieldintermediate 14 (0.687 g, 63%) as a colorless oil.

Method 2: Intermediate 13 (obtained from method 2, 0.172 g, 0.912 mmol)was added to a stirred solution of NaH (60% dispersion in mineral oil,0.055 g, 1.368 mmol) in DMF (7.3 mL) at 0° C. under nitrogen atmosphere.After being stirred for 30 min at this temperature, CH₃I (0.107 mL,1.459 mmol) was added and stirred overnight at rt. The mixture wasdiluted with water and extracted with EtOAc. The organic layer wasfiltered, dried and evaporated in vacuo. The crude product was purifiedby flash column chromatography (EtOAc in heptane, 0/100 to 10/90). Thedesired fractions were collected and concentrated in vacuo to yieldintermediate 14 (0.143 g, 77%).

Preparation of Intermediate 15

2-Chloro-5-hydroxypyridine (0.300 g 2.316 mmol) and2-fluoroethyl-4-methylbenzenesulfonate (0.505 g, 2.316 mmol, CAS383-50-6) were dissolved in DMF (7 mL). Cs₂CO₃ (1.132 g, 3.474 mmol) wasadded under nitrogen atmosphere. The mixture was heated to 85° C. for 5h. After cooling to ambient temperature, the dark brown suspension wasdiluted with water and extracted with EtOAc. The org layers were washedwith brine, dried over MgSO₄, filtered and concentrated. The product waspurified by flash column chromatography (silica; EtOAc in heptane 0/100to 35/65). The desired fractions were collected and concentrated invacuo providing intermediate 15 (0.374 g, 92%).

Preparation of Intermediate 16

2-Chloro-5-hydroxypyridine (0.300 g, 2.316 mmol) and 3-fluoropropyl4-methylbenzenesulfonate (0.538 g, 2.316 mmol, CAS 312-68-5) weredissolved in DMF (7 mL). Cs₂CO₃ (1.132 g, 3.474 mmol) was added undernitrogen atmosphere. The mixture was heated to 85° C. for 5 h. Aftercooling to ambient temperature, the dark brown suspension was dilutedwith water and extracted with EtOAc. The organic layer was washed withbrine, dried over MgSO₄, filtered and concentrated. The product waspurified by flash column chromatography (silica; EtOAc in heptane, 0/100to 30/70). The desired fractions were collected and concentrated invacuo providing intermediate 16 (0.408 g, 93%).

Preparation of Intermediate 17

K₃PO₄ (1.70 g, 7.99 mmol), Pd₂(dba)₃ (0.152 g, 0.166 mmol) and Xantphos(0.161 mg, 0.277 mmol) were added to a solution of 2,5-dibromopyridine(0.738 g, 3.052 mmol, CAS 624-28-2) in dry THF (20 mL) while nitrogenwas bubbling through the mixture. After 10 min, 6-aminoisoquinoline(0.400 g, 2.774 mmol, CAS 23687-26-5) was added and the mixture wasstirred at rt for 10 min. Then, the mixture was heated at 100° C. for 16h. After cooling to rt, the mixture was washed with aq. sat. NaHCO₃ andextracted with EtOAc. The organic layer was dried over MgSO₄, filteredand the solvent was evaporated in vacuo. The crude product was purifiedby flash column chromatography (silica; EtOAc in heptane 0/100 to75/25). The desired fractions were collected and concentrated in vacuoproviding intermediate 17 (0.680 g, 80% pure, 65% yield).

Preparation of Intermediate 18

Intermediate 17 (0.680 g, 1.812 mmol) in 1,4-dioxane (10.2 mL) wasdegassed for 10 min.2-[2-Ethoxyvinyl]-4,4,5,5-tetramethyl-1,3,2-dioxaborolane (0.538 g,2.719 mmol, CAS 1201905-61-4, prepared according to procedure describedin WO2012010538, 2:1 mixture of E and Z), Pd(OAc)₂ (0.020 g 0.0906mmol), X-Phos (0.095 g, 0.199 mmol) and Cs₂CO₃ (0.886 g, 2.719 mmol)were added followed by degassed water (1.1 mL). The mixture was degassedfor another 10 min and was stirred at 80° C. for 6 h. The mixture wasthen cooled to ambient temperature and diluted with EtOAc. The org phasewas washed with water and brine, dried over MgSO₄, filtered andconcentrated. The crude product was purified by flash columnchromatography (silica; EtOAc in heptane 0/100 to 100/0). The desiredfractions were collected and concentrated in vacuo providingintermediate 18 (0.510 g, 91%, mixture of E and Z).

Preparation of Intermediate 19

To a mixture of intermediate 18 (0.510 g, 1.75 mmol) in THF (5.3 mL) wasadded HCl (2 M in water, 3.939 mL, 7.877 mmol). The mixture was stirredat 60° C. overnight. NaHCO₃ sat. was added until pH 7. The mixture wasextracted with EtOAc. The org layer was separated, dried (MgSO₄),filtered and the solvents were evaporated in vacuo providingintermediate 19. The product was used as such in the next step (0.378 g,82%).

Preparation of Intermediate 20

NaBH₄ (0.054 g, 1.436 mmol) was added to a solution of intermediate 19in MeOH (4.5 mL) at 0° C. The mixture was stirred at rt for 30 min.Water was added and the mixture was extracted with EtOAc. The org layerwas separated, dried (MgSO₄), filtered and the solvents were evaporatedin vacuo. The crude product was purified by flash column chromatography(silica; MeOH in DCM 0/100 to 10/90). The desired fractions werecollected and concentrated in vacuo providing intermediate 20 (0.135 g,35%).

B. Preparation of Compounds of Formula [¹⁹F](I)

Preparation of Compound 1

Method 1: K₃PO₄ (0.872 g, 4.11 mmol), Pd₂(dba)₃ (0.078 g, 0.0856 mmol)and Xantphos (0.083 g, 0.143 mmol) were added to a solution of6-bromoisoquinoline (0.327 g, 1.57 mmol) in dry DMF (15 mL) whilenitrogen was bubbled through the mixture. After 10 min, intermediate 1(0.180 g, 1.427 mmol) was added and the mixture was stirred at rt for 10min. Then, the mixture was heated at 100° C. for 16 h. After cooling tort, the mixture was washed with sat. NaHCO₃ and extracted with EtOAc.The org layer was dried over MgSO₄, filtered and the solvent wasevaporated in vacuo. The crude product was purified by flash columnchromatography (silica; DCM/MeOH, from 100/0 to 95/5). The desiredfractions were collected and concentrated in vacuo. The product wasfurther purified by reverse phase from 70% H₂O (25 mM NH₄HCO₃)—30%MeCN-MeOH to 27% H₂O (25 mM NH₄HCO₃)—73% MeCN-MeOH. The desiredfractions were collected and concentrated in vacuo. The product wastriturated with DIPE to yield compound 1 as a white solid (0.150 g,41%).

Method 2: KF (10.2 g, 175.7 mmol) was added to intermediate 2 (obtainedfrom method 2. 9.85 g, 35.14 mmol) in DMSO (236 mL). The resultingmixture was stirred for 16 h at 160° C., after which LCMS showed fullconversion. Water and DCM were added, the biphasic mixture was shaken,and then filtered over Dicalite®. The org. layer was separated and theaq. layer extracted with DCM. The combined org layers were dried(MgSO₄), filtered and evaporated to dryness. The red-brown residue waspurified by flash column chromatography over 120 g silica gel using agradient (heptane/EtOAc, 1:0 to 0:1). The product fractions wereevaporated to dryness providing a brown solid. A purification wasperformed via Prep HPLC (Stationary phase: RP XBridge Prep C18 OBD-10μm, 50×150 mm, Mobile phase: 0.25% NH₄HCO₃ solution in water, MeOH)yielding compound 1 as white crystals (1.38 g, 15.5%) after evaporationof the eluent, suspending in water, filtering, washing with heptane, anddrying in vacuo. ¹H NMR (400 MHz, DMSO-d₆) δ ppm 2.17 (s, 3H) 6.75 (d,J=11.9 Hz, 1H) 7.62 (d, J=5.7 Hz, 1H) 7.66 (dd, J=8.9, 2.1 Hz, 1H) 7.97(d, J=8.8 Hz, 1H) 8.20 (d, J=11.2 Hz, 1H) 8.33 (d, J=5.7 Hz, 1H) 8.42(d, J=1.8 Hz, 1H) 9.06 (s, 1H) 9.61 (s, 1H)

Preparation of Compound 2

K₃PO₄ (0.462 g, 2.175 mmol), Pd₂(dba)₃ (0.041 g, 0.0453 mmol) andXantphos (0.044 g, 0.0755 mmol) were added to a solution of intermediate3 (0.100 g, 0.793 mmol) in dry THF while nitrogen was bubbling throughthe mixture. After 10 min, 6-bromoisoquinoline (0.157 g, 0.755 mmol) wasadded and the mixture was stirred at rt for 10 min. Then, the mixturewas heated at 100° C. for 16 h. After cooling to rt, the mixture waswashed with aq. sat. NaHCO₃ and extracted with EtOAc. The organic layerwas dried over MgSO₄, filtered and the solvent was evaporated in vacuo.The crude product was purified by flash column chromatography (silica;DCM/MeOH, 100/0 to 95/5). The desired fractions were collected andconcentrated in vacuo. The residue was purified by preparative HPLC((0.1% HCOOH)/25 mM NH₄HCO₃); from 70/30 to 27/73). The desiredfractions were collected and concentrated in vacuo yielding compound 2(0.0238 g, 12%). ¹H NMR (300 MHz, CHLOROFORM-d) δ ppm 2.24 (s, 3H)6.72-6.86 (m, 2H) 7.40-7.60 (m, 3H) 7.89 (d, J=8.7 Hz, 1H) 7.93 (br s,1H) 8.44 (d, J=5.8 Hz, 1H) 9.10 (br s, 1H)

Preparation of Compound 3

K₃PO₄ (0.300 g, 1.415 mmol), Pd₂(dba)₃ (0.027 g, 0.0295 mmol) andXantphos (0.028 g, 0.0491 mmol) were added to a solution of intermediate4 (0.080 g, 0.491 mmol) in dry THF (5 mL) while nitrogen was bubblingthrough the mixture. After 10 min, isoquinolin-6-amine (0.074 g, 0.516mmol, CAS 23687-26-5) was added and the mixture was stirred at rt for 10min. Then, the mixture was heated at 90° C. for 16 h. After cooling tort, the mixture was washed with sat. NaHCO₃ and extracted with EtOAc.The org layer was dried over MgSO₄, filtered and the solvent wasevaporated in vacuo. The crude product was purified by flash columnchromatography (silica; MeOH in DCM, 0/100 to 3/97). The desiredfractions were collected and concentrated in vacuo. The product wasfurther purified by preparative HPLC ((0.1% HCOOH)/ACN: MeOH 1:1); from95/5 to 63/37). The desired fractions were collected, concentrated invacuo to yield compound 3 (0.051 g, 38%). ¹H NMR (300 MHz, CHLOROFORM-d)δ ppm 2.98 (dt, J=24.7, 6.2 Hz, 2H) 4.64 (dt, J=47.0, 6.3 Hz, 2H) 6.82(br s, 1H) 6.98 (d, J=8.4 Hz, 1H) 7.43-7.56 (m, 3H) 7.89 (d, J=8.8 Hz,1H) 7.98 (s, 1H) 8.22 (s, 1H) 8.43 (d, J=5.8 Hz, 1H) 9.10 (s, 1H).

Compound 3 can be also made alternatively from intermediate 20, forexample, by formation of the corresponding methanesulfonate or4-toluenesulfonate followed by displacement with a source of fluoride.

Preparation of Compound 4

K₃PO₄ (0.481 g, 2.264 mmol), Pd₂(dba)₃ (0.043 g, 0.0472 mmol) andXantphos (0.045 g, 0.0786 mmol) were added to a solution of intermediate6 (0.127 g, 0.786 mmol) in THF (6 mL) while nitrogen was bubbling. After10 min, isoquinolin-6-amine (0.125 g, 0.865 mmol, CAS 23687-26-5) wasadded and the mixture was stirred at rt for 10 min. Then, the mixturewas heated at 90° C. for 16 h. After cooling to rt, the mixture waswashed with sat. NaHCO₃ and extracted with EtOAc. The organic layer wasdried over MgSO₄, filtered and the solvent was evaporated in vacuo. Thecrude product was purified by flash column chromatography (silica; EtOAcin heptane from 5/95 to 100/0). The desired fractions were collected andconcentrated in vacuo. The product was purified by preparative HPLC((from 59% H₂O (25 mM NH₄HCO₃)—41% MeCN-MeOH to 17% H₂O (25 mMNH₄HCO₃)—83% MeCN-MeOH). The desired fractions were collected andconcentrated in vacuo. The product was triturated with n-pentane toyield compound 4 as a beige solid (0.075 g, 34%). ¹H NMR (300 MHz,CHLOROFORM-d) δ ppm 5.69 (d, J=54.6 Hz, 2H) 6.80 (br s, 1H) 7.00 (d,J=8.9 Hz, 1H) 7.39-7.47 (m, 1H) 7.54 (d, J=5.8 Hz, 1H) 7.89 (d, J=8.8Hz, 1H) 7.97 (s, 1H) 8.22 (d, J=2.1 Hz, 1H) 8.43 (d, J=5.8 Hz, 1H) 9.09(s, 1H)

Preparation of Compound 5

K₃PO₄ (0.294 g, 1.386 mmol), Pd₂(dba)₃ (0.026 g, 0.0289 mmol) andXantphos (0.028 g, 0.0481 mmol) were added to a solution of intermediate10 (0.084 g, 0.481 mmol) in THF (4 mL) while nitrogen was bubbling.After 10 min, isoquinolin-6-amine (0.069 g 0.481 mmol) was added and themixture was stirred at rt for 10 min. Then, the mixture was heated at100° C. for 16 h. Pd₂(dba)₃ (0.026 g, 0.0289 mmol) and Xantphos (0.028g, 0.0481 mmol) were added while nitrogen was bubbling, and the mixturewas further heated at 100° C. overnight. Pd₂(dba)₃ (0.026 g, 0.0289mmol) and Xantphos (0.028 g, 0.0481 mmol) were added while nitrogen wasbubbling, and the mixture was further heated at 100° C. overnight. Themixture was washed with aq. sat. NaHCO₃ and extracted with EtOAc. Theorganic layer was dried over MgSO₄, filtered and the solvent wasevaporated in vacuo. The crude product was purified by flash columnchromatography (silica; EtOAc in DCM 0/100 to 50/50). The desiredfractions were collected and concentrated in vacuo. The product wasfurther purified by preparative HPLC (from 75% [25 mM NH₄HCO₃]—25% [ACN:MeOH 1:1] to 38% [25 mM NH₄HCO₃]—62% [ACN: MeOH 1:1]). The desiredfractions were collected and the solvent was evaporated. The product wastriturated with Et₂O and filtrated to yield compound 5 (0.042 g, 31%).¹H NMR (300 MHz, DMSO-d₆) δ ppm 3.34-3.48 (m, 2H) 4.58 (dt, J=47.6, 4.8Hz, 2H) 5.63 (br t, J=5.9 Hz, 1H) 6.86 (d, J=8.8 Hz, 1H) 7.11 (dd,J=8.8, 2.7 Hz, 1H) 7.47-7.57 (m, 2H) 7.78 (d, J=2.5 Hz, 1H) 7.87 (d,J=8.9 Hz, 1H) 8.21-8.31 (m, 2H) 8.96 (s, 1H) 9.12 (s, 1H)

Preparation of Compound 6

K₃PO₄ (0.444 g, 2.092 mmol), Pd₂(dba)₃ (0.040 g, 0.0436 mmol) andXantphos (0.042 g, 0.0726 mmol) were added to a solution of intermediate11 (0.137 g, 0.726 mmol) in THF (6 mL) while nitrogen was bubbling.After 10 min, isoquinolin-6-amine (0.105 g, 0.726 mmol) was added andthe mixture was stirred at rt for 10 min. Then, the mixture was heatedat 100° C. for 16 h. More Pd₂(dba)₃ (0.026 g, 0.0289 mmol) and Xantphos(0.028 g, 0.0481 mmol) were added while nitrogen was bubbling throughthe mixture, after which it was further heated at 100° C. overnight. Themixture was washed with aq. sat. NaHCO₃ and extracted with EtOAc. Thecombined organic layers were dried over MgSO₄, filtered and the solventwas evaporated in vacuo. The crude product was purified by flash columnchromatography (silica; EtOAc in DCM 0/100 to 65/35). The desiredfractions were collected and concentrated in vacuo. The product wasfurther purified by preparative HPLC (from 70% [25 mM NH₄HCO₃]—30% [ACN:MeOH 1:1] to 27% [25 mM NH₄HCO₃]—73% [ACN: MeOH 1:1]). The desiredfractions were collected and the solvent was evaporated. The product wastriturated with Et₂O and filtrated to yield compound 6 (0.092 g, 43%).¹H NMR (300 MHz, DMSO-d₆) δ ppm 2.93 (s, 3H) 3.62 (dt, J=26.7, 4.6 Hz,2H) 4.60 (dt, J=47.7, 4.7 Hz, 2H) 6.93 (d, J=9.1 Hz, 1H) 7.28 (dd,J=9.0, 2.7 Hz, 1H) 7.46-7.60 (m, 2H) 7.82-7.94 (m, 2H) 8.27 (d, J=5.8Hz, 1H) 8.34 (s, 1H) 8.97 (s, 1H) 9.21 (s, 1H)

Preparation of Compound 7

K₃PO₄ (0.544 g, 2.565 mmol), Pd₂(dba)₃ (0.049 g, 0.0534 mmol) andXantphos (0.052 g, 0.0891 mmol) were added to a solution of intermediate13 (0.168 g, 0.891 mmol) in THF (6 mL) while nitrogen was bubbling.After 10 min, isoquinolin-6-amine (0.128 g, 0.891 mmol) was added andthe mixture was stirred at rt for 10 min. Then, the mixture was heatedat 100° C. for 16 h. More Pd₂(dba)₃ (0.049 g, 0.0534 mmol) and Xantphos(0.052 g, 0.0891 mmol) were added under nitrogen flow, and the mixturewas heated again at 100° C. overnight. Pd₂(dba)₃ (0.049 g, 0.0534 mmol)and Xantphos (0.052 g, 0.0891 mmol) were added were added under nitrogenflow, and the mixture was heated again at 100° C. for 6 h. The mixturewas washed with sat. NaHCO₃ and extracted with EtOAc. The org layerswere dried over MgSO₄, filtered and the solvent was evaporated in vacuo.The crude product was purified by flash column chromatography (silica;EtOAc in DCM 0/100 to 45/55). The desired fractions were collected andconcentrated in vacuo. The product was purified by preparative HPLC(from 75% [25 mM NH₄HCO₃]—25% [ACN: MeOH 1:1] to 0% [25 mM NH₄HCO₃]—100%[ACN: MeOH 1:1]). The desired fractions were collected and the solventwas evaporated. The residue was dissolved in DCM and HCl (5 M in2-propanol) was added and the resulting mixture was concentrated invacuo. The residue was triturated with Et₂O and filtered to yieldcompound 7 as an orange solid (0.066 g, 22% yield). ¹H NMR (300 MHz,DMSO-d₆) δ ppm 1.83-2.05 (m, 2H) 3.17 (br t, J=6.8 Hz, 2H) 4.58 (dt,J=47.4, 5.8 Hz, 2H) 5.83 (br s, 1H) 7.00 (d, J=8.8 Hz, 1H) 7.12 (dd,J=8.7, 2.7 Hz, 1H) 7.77 (dd, J=9.2, 1.2 Hz, 1H) 7.81 (d, J=2.5 Hz, 1H)7.99 (d, J=6.9 Hz, 1H) 8.20 (d, J=9.1 Hz, 1H) 8.28 (d, J=6.6 Hz, 1H)8.42 (s, 1H) 9.30 (s, 1H) 10.08 (s, 1H)

Preparation of Compound 8

K₃PO₄ (0.431 g, 2.032 mmol), Pd₂(dba)₃ (0.039 g, 0.0423 mmol) andXantphos (0.041 g, 0.0706 mmol) were added to a solution of intermediate14 (0.143 g, 0.706 mmol) in THF (5.9 mL) while nitrogen was bubblingthrough the mixture. After 10 min, isoquinolin-6-amine (0.102 g, 0.706mmol) was added and the mixture was stirred at rt for 10 min. Then, themixture was heated at 100° C. for 16 h. Additional Pd₂(dba)₃ (0.039 g,0.0423 mmol) and Xantphos (0.041 g, 0.0706 mmol) were added whilenitrogen was bubbling through the mixture, after which it was heated at100° C. overnight. Additional Pd₂(dba)₃ (0.039 g, 0.0423 mmol) andXantphos (0.041 g, 0.0706 mmol) were added while nitrogen was bubbling,and the mixture was heated at 100° C. for 6 h. The mixture was washedwith sat. NaHCO₃ and extracted with EtOAc. The org layer was dried overMgSO₄, filtered and the solvent was evaporated in vacuo. The crudeproduct was purified by flash column chromatography (silica; EtOAc inDCM 0/100 to 20/80). The desired fractions were collected andconcentrated in vacuo. The product was further purified by preparativeHPLC (from 75% [25 mM NH₄HCO₃]-25% [ACN: MeOH 1:1] to 0% [25 mMNH₄HCO₃]—100% [ACN: MeOH 1:1]). The desired fractions were collected andthe solvent was evaporated. The crude was dissolved in DCM, HCl (5 N in2-propanol) was added and the mixture was concentrated in vacuo. Theresidue was triturated with Et₂O and filtered to yield compound 8 as anorange solid (0.058 g, 23%). ¹H NMR (300 MHz, DMSO-d₆) δ ppm 1.78-2.02(m, 2H) 2.92 (s, 3H) 3.45 (br t, J=7.1 Hz, 2H) 4.52 (dt, J=47.4, 5.7 Hz,2H) 7.09 (d, J=8.9 Hz, 1H) 7.29 (dd, J=9.0, 3.0 Hz, 1H) 7.81 (dd, J=8.8,1.2 Hz, 1H) 7.91 (d, J=2.6 Hz, 1H) 7.98 (d, J=6.6 Hz, 1H) 8.21 (d, J=9.1Hz, 1H) 8.29 (d, J=6.7 Hz, 1H) 8.51 (s, 1H) 9.32 (s, 1H) 10.20 (s, 1H)

Preparation of Compound 9

K₃PO₄ (0.449 g, 2.117 mmol), Pd₂(dba)₃ (0.040 g, 0.0441 mmol) andXantphos (0.043 g, 0.0735 mmol) were added to a solution of intermediate15 (0.142 g, 0.809 mmol) in THF (4 mL) while nitrogen was bubblingthrough the mixture. After 10 min, isoquinolin-6-amine (0.106 g, 0.735mmol) was added and the mixture was stirred at rt for 10 min. Then, themixture was heated at 100° C. for 16 h. After cooling to rt, the mixturewas washed with aq. sat. NaHCO₃ and extracted with EtOAc. The combinedorganic layers were dried over MgSO₄, filtered and the solvent wasevaporated in vacuo. The crude product was purified by flash columnchromatography (silica gel; EtOAc in Heptane, 0/100 to 40/60). Thedesired fractions were collected and concentrated in vacuo. The productwas triturated with di-isopropyl ether and filtered to yield compound 9as a beige solid (0.116 g, 55%). ¹H NMR (300 MHz, DMSO-d₆) δ ppm 4.28(br dt, J=30.2, 3.3 Hz, 2H) 4.75 (br dt, J=47.9, 3.3 Hz, 2H) 6.99 (d,J=8.9 Hz, 1H) 7.43 (dd, J=8.9, 2.7 Hz, 1H) 7.52-7.68 (m, 2H) 7.93 (d,J=8.8 Hz, 1H) 8.07 (d, J=2.5 Hz, 1H) 8.30 (br d, J=5.6 Hz, 1H) 8.42 (s,1H) 9.02 (s, 1H) 9.44 (s, 1H)

Preparation of Compound 10

K₃PO₄ (0.449 g, 2.117 mmol), Pd₂(dba)₃ (0.040 g 0.0441 mmol) andXantphos (0.043 g, 0.0735 mmol) were added to a solution of intermediate16 (0.153 g, 0.809 mmol) in THF (4 mL) while nitrogen was bubblingthrough the mixture. After 10 min, isoquinolin-6-amine (0.106 g, 0.735mmol) was added and the mixture was stirred at rt for 10 min. Then, themixture was heated at 100° C. for 16 h. The mixture was washed with aq.sat. NaHCO₃ and extracted with EtOAc. The combined org layers were driedover MgSO₄, filtered and the solvent was evaporated in vacuo. The crudeproduct was purified by flash column chromatography; (silica; EtOAc inHeptane, 0/100 to 35/65). The desired fractions were collected andconcentrated in vacuo. The residue was dissolved in DCM, HCl (5 N in2-propanol) was added and the mixture was concentrated in vacuo. Theresidue was triturated with DIPE and filtered to yield compound 10 as ayellow solid (0.147 g, 47%). ¹H NMR (300 MHz, DMSO-d₆) δ ppm 2.00-2.23(m, 2H) 4.16 (t, J=6.2 Hz, 2H) 4.63 (dt, J=47.3, 5.8 Hz, 2H) 7.18 (d,J=8.9 Hz, 1H) 7.51 (dd, J=8.9, 3.0 Hz, 1H) 7.91 (dd, J=9.2, 1.4 Hz, 1H)8.07 (d, J=6.7 Hz, 1H) 8.13 (d, J=2.7 Hz, 1H) 8.28 (d, J=9.1 Hz, 1H)8.34 (d, J=6.6 Hz, 1H) 8.63 (s, 1H) 9.41 (s, 1H) 10.44 (s, 1H)

C. Radiosynthesis

Materials and Methods

General

The nonradioactive reference material for T808 was synthesized byJanssen Research & Development (a division of Janssen Pharmaceutica NV,Beerse, Belgium) following literature reports (Declercq L., Celen S.,Lecina J. et al. Molecular Imaging 2016, 15, 1-151). All chemicals andreagents were purchased from commercial sources and used without furtherpurification. HPLC analysis was performed on a LaChrom Elite HPLC system(Hitachi, Darmstadt, Germany) connected to a UV detector set at 254 nm.For analysis of radiolabeled compounds, the HPLC eluate, after passingthrough the UV-detector, was led over a 3-inch NaI (TI) scintillationdetector connected to a single channel analyzer (GABI box; Raytest,Straubenhardt, Germany). Data were acquired and analyzed using GINA Star(Raytest) data acquisition systems.

Tritiation Co. No. 1

Co. No. 1 (1.74 mg, 6.87 μmol) and(1,2,5,6-η)-1,5-cyclooctadiene](pyridine)(tricyclohexylphosphine)iridium(I) hexafluorophosphate, also referred toas Crabtree's catalyst (7.72 mg, 9.59 μmol) was dissolved in CH₂Cl₂ (0.6mL). The bright orange solution was degassed three times on an RC Tritectritiation manifold and then stirred under an atmosphere of tritium gas(8.9 Ci) for 3.5 h at room temperature. The maximum pressure reachedduring the reaction was 882 mbar. After reaction, the solvent wasremoved under vacuo. Labile tritium was exchanged by adding methanol(0.8 mL), stirring the solution, and removing the solvent again undervacuo. This process was repeated three times to afford a dried solidcrude product.

The crude product was purified by HPLC method: Macherey+Nagel NucleodurGravity C18, 5 μm, 8×150 mm column; mobile phase A: water with 0.1% TFA,B: acetonitrile with 0.1% TFA; isocratic at 27% B; flow rate 3.1 mL/min,230 nm and 254 nm, 20° C. After HPLC, the pH of the collected productfractions was set to neutral with an aqueous solution of NaHCO₃ (10%).The volume of the mixture was reduced on a rotary evaporator. Then theproduct was extracted with a Phenomenex StrataX cartridge (3 mL, 100mg), which was eluted with ethanol (5 mL). The product received wasfurther purified by HPLC under basic conditions: Macherey+NagelNucleodur Sphinx RP, 5 μm, 8×150 mm; mobile phase A: 10 mM NH₄OAc with0.05% NH₄OH, B: acetonitrile; isocratic at 46.5% B; flow rate 3.1mL/min, 230 nm and 254 nm, 20° C. The volume of the collected productfractions was reduced on a rotary evaporator. Then the product wasextracted with a Phenomenex StrataX cartridge (3 mL, 100 mg), which waseluted with ethanol (5 mL).

The radiochemical purity of [³H] Co. No. 1 was determined to be >99% bythe following HPLC method: Column: Macherey+Nagel Nucleodur Sphinx RP (5μm), 4.6×150 mm, Column temperature: 30° C., Mobile phase A: 10 mMNH₄OAc with 0.05% v/v NH₄OH in water, Mobile phase B: Acetonitrile, Flowrate: 1.0 mL/min, Injection volume: 5 μL, Detection wavelength: UV at254 nm, Elution gradient: Gradient from 5% B to 95% B in 0-20 min;isocratic at 95% B in 20-25 min; gradient from 95% B to 5% B in 25-25.5min. The radioactivity flow detector was a Berthold LB 513 with ZinsserQuickszint Flow 302 cocktail at a flow rate of 3.0 mL/min.

The specific activity was determined to 57.7 mCi/mmol by massspectrometry. LCMS conditions: Agilent Zorbax SB C18 (1.8 μm) 2.1×50 mmcolumn; Mobile phase A: water 0.1% formic acid, B: MeCN 0.1% formicacid; 0 min 5% B; 0.2 min 5% B; 5 min 80% B; Flow rate 0.6 mL/min;Injection 1.0 μL (1.06 μCi, 39.2 KBq), UV-detection 225 nm; Temperature60° C.

Radiofluorination Co. No. 1 (N=6)

Fluoride-18 ([¹⁸F]F⁻) was produced by an ¹⁸O(p,n)¹⁸F nuclear reaction ina Cyclone 18/9 cyclotron (Ion Beam Applications, Louvain-la-Neuve,Belgium) by irradiation of 2 mL of 97% enriched ¹⁸O—H₂O (Rotem HYOX18,Rotem Industries, Beer Sheva, Israel) with 18-MeV protons. Afterirradiation, [¹⁸F]F⁻ was trapped on a SepPak Light Accell plus QMA anionexchange cartridge (CO₃ ²⁻ form, Waters, Milford, Mass., U.S.A.) andeluted with a mixture of Kryptofix 2.2.2 (K-222, 27.86 mg) and K₂CO₃(2.46 mg) dissolved in CH₃CN/H₂O (0.75 mL; 95:5 v/v). After evaporationof the solvent with a stream of helium at 80° C. and 35 W (microwavecavity), anhydrous CH₃CN (1 mL) was added, and [¹⁸F]F⁻ was further driedunder the same conditions. A solution of 0.50 mg of the nitro precursorin 0.25 mL DMSO was added to the dried [¹⁸F]F⁻/K₂CO₃/K-222 residue andthe mixture was heated at 160° C. and 50 W for 10 min in a microwavecavity. The crude radiolabelling mixture was diluted with 0.6 mLpreparative buffer (0.01 M Na₂HPO₄ pH 7.4 and EtOH (60:40 v/v)) andpurified using reverse phase HPLC (RP-HPLC) on an XBridge C₁₈ column (5μm, 4.6 mm×150 mm; Waters, Milford, U.S.A.) eluted with a mixture ofNa₂HPO₄ pH 7.4 and EtOH (60:40 v/v) at a flow rate of 0.8 mL/min andwith UV detection at 254 nm. [¹⁸F]Co. No. 1 eluted at 27 min. Thepurified radiotracer solution was diluted with saline to obtain anethanol concentration <10%, suitable for intravenous injection. Thesolution was subsequently passed through a 0.22-μm filter (Millex-GV,Millipore, Billerica, Mass., U.S.A.) to obtain a sterile product.Quality control was performed using RP-HPLC on an XBridge column (C₁₈,3.5 μm, 3.0 mm×100 mm; Waters, Milford, U.S.A.) eluted with a mixture of0.01 M Na₂HPO₄ pH 7.4 and CH₃N (68:32 v/v) at a flow rate of 0.8 mL/min.UV detection was performed at 254 nm. [¹⁸F]Co. No. 1 eluted at 8 min.[¹⁸F]Co. No. 1 was obtained with an average, decay-corrected,radiochemical yield of 12% respectively (relative to radioactivity of[¹⁸F]F⁻ in the preparative chromatogram,n=6). Their radiochemical puritywas examined using HPLC on an analytical C₁₈ column and was more than99%. [¹⁸F]Co. No. 1 was obtained within a total synthesis time of 60min, and collected with an average specific radioactivity of 85 GBq/μmolat the end of synthesis (EOS, n=6).

D. Analytical Part

Melting Points

Values are either peak values or melt ranges, and are obtained withexperimental uncertainties that are commonly associated with thisanalytical method.

For a number of compounds, melting points were determined with a DSC823e(Mettler-Toledo) (indicated as DSC). Melting points were measured with atemperature gradient of 10° C./minute. Maximum temperature was 300° C.

For a number of compounds, melting points were determined in opencapillary tubes on a Mettler Toledo MP50. Melting points were measuredwith a temperature gradient of 10° C./minute. Maximum temperature was300° C. The melting point data was read from a digital display andchecked from a video recording system.

LC/MS Methods

The High Performance Liquid Chromatography (HPLC) measurement wasperformed using a LC pump, a diode-array (DAD) or a UV detector and acolumn as specified in the respective methods. If necessary, additionaldetectors were included (see table of methods below).

Flow from the column was brought to the Mass Spectrometer (MS) which wasconfigured with an atmospheric pressure ion source. It is within theknowledge of the skilled person to set the tune parameters (e.g.scanning range, dwell time . . . ) in order to obtain ions allowing theidentification of the compound's nominal monoisotopic molecular weight(MW). Data acquisition was performed with appropriate software.Compounds are described by their experimental retention times (R_(t))and ions. If not specified differently in the table of data, thereported molecular ion corresponds to the [M+H]⁺ (protonated molecule)and/or [M−H]⁻ (deprotonated molecule). In case the compound was notdirectly ionizable the type of adduct is specified (i.e. [M+NH₄]⁺,[M+HCOO]⁻, etc. . . . ). For molecules with multiple isotopic patterns(Br, Cl), the reported value is the one obtained for the lowest isotopemass. All results were obtained with experimental uncertainties that arecommonly associated with the method used. Hereinafter, “SQD” meansSingle Quadrupole Detector, “MSD” Mass Selective Detector, “RT” roomtemperature, “BEH” bridged ethylsiloxane/silica hybrid, “DAD” DiodeArray Detector, “HSS” High Strength silica.

TABLE 1a LCMS Method codes (Flow expressed in mL/min; column temperature(T) in ° C.; Run time in minutes) Flow Run Method ------- time No.Instrument Column Mobile phase Gradient Col T (min) 1 Waters: Waters: A:10 mM From 100% A to 0.7 3.5 Acquity ® HSS T3 CH₃COONH₄ 5% A in -------UPLC ®-DAD (1.8 μm, in 95% H₂O + 2.10 min, 55 and SQD 2.1*100 5% CH₃CNto 0% A in mm) B: CH₃CN 0.90 min, to 5% A in 0.5 min 2 Waters: Waters:A: 10 mM From 95% A to 0.8 2 Acquity ® BEH C18 CH₃COONH⁴ 5% A in 1.3min, ------- UPLC ®- (1.7 μm, in 95% H₂O + held for 0.7 min 55 DAD andSQD 2.1*50 mm) 5% CH₃CN B: CH₃CN 3 Agilent: 1100- YMC: Pack A: HCOOH 95%A to 5% A 2.6 6 DAD and MSD ODS-AQ 0.1% in water, in 4.8 min, held (3μm, B: CH₃CN for l min, back 4.6 × 50 mm) to 95% A in 0.2 min. 4 Agilent1290 YMC-pack A: 0.1% ISET 2V1.0 2.6 6.0 Infinity DAD ODS-AQ HCOOH inEmulated ------- TOF-LC/MS C18 (50 × H₂O Agilent Pump 35 G6224A 4.6 mm,B: CH₃CN G1312A V1.0 3 μm) From 94.51% A to 5% A in 4.8 min, held for1.0 min, to 95% A in 0.2 min 5 Waters: Waters : A: 10 mM From 95% A toAcquity ® BEH C18 CH₃COONH₄ 5% A in 1.3 0.7 1.8 UPLC ®- (1.7 μm, in 95%H₂O + min, held for 0.2 ------- DAD and SQD 2.1*50 mm) 5% CH₃CN min, to95% A 70 B: CH3CN in 0.2 min held for 0.1 min

TABLE 1b Analytical LCMS data—R_(t) means retention time (in minutes),[M + H]⁺ means the protonated mass of the compound, method refers to themethod used for (LC)MS analysis. Co. LCMS No. mp (° C.) Rt [M + H]⁺ [M −H]⁻ Method 1-2 0.94 281.2 279.1 2 1 182.53° C.  1.84 254.2 252.1 1(DSC)* 2 203.3° C. 2.073 254.0 3 3 208.5° C. 1.878 268.0 3 4 173.1° C.1.772 270.0 3 5 191.4° C. 1.413 283.2 4 6 146.3° C. 1.647 297.2 4 7285.1° C. 1.533 297.2 4 8 1.758 311.2 4 9 104.6° C. 1.72 284.2 282.2 510 249.3° C. 1.947 298.2 4 *from 30 to 300° C. at 10° C./min 50 mL N₂

CHN Determinations & Water Determinations

For a number of compounds, amount of Carbon, Hydrogen and Nitrogen (CHN)in (% w/w) was determined by Dynamic Flash Combustion on an EA 1108 CHNanalyzer from Fisons instruments.

For a number of compounds, water (% w/w) was determined with aCA-02-moisture meter (Mitsubishi) Coulometric principle applied to KarlFischer titration.

TABLE 1c CHN and water determinations. Co. No. C (% w/w) H (% w/w) N (%w/w) H₂O (% w/w) 1 70.52 4.95 16.79 1-2 57.63 3.96 15.07 0.42

E. Biological Part

Materials and Methods

General

Quantification of radioactivity in samples of biodistribution andradiometabolite studies was performed using an automated γ-counterequipped with a 3-inch NaI(Tl) well crystal coupled to a multichannelanalyzer, mounted in a sample changer (Wallac 2480 Wizard 3q, Wallac,Turku, Finland). The values are corrected for background radiation,physical decay and counter dead time. Rodents were housed inindividually ventilated cages in a thermo-regulated (˜22° C.),humidity-controlled facility under a 12 h-12 h light-dark cycle, withaccess to food and water ad libitum. All animal experiments wereconducted according to the Belgian code of practice for the care and theuse of animals, after approval from the university animal ethicscommittee.

Aggregated Tau and Amyloid Plaques Isolation from Human AD Brain

Enriched aggregated tau fractions were prepared according to a slightlymodified version of the protocol described by Greenberg and Davies(Greenberg S. G., Davies P. A preparation of Alzheimer paired helicalfilaments that displays distinct

proteins by polyacrylamide gel electrophoresis. Proc. Natl. Acad. Sci.1990; 87: 5827-5831) using human AD brain tissue (occipital cortex withhigh tau fibril load). Briefly, frozen human AD brain samples (˜10 g)were homogenized with 10 vol of cold homogenization buffer (10 mM Tris,800 mM NaCl, 1 mM EGTA, 10% sucrose, pH 7.4 containing PhosSTOPphosphatase and cOmplete EDTA-free protease inhibitor (Roche, Vilvoorde,Belgium)) on ice. After centrifugation at 27 000×g for 20 min at 4° C.the supernatant was recovered and 1% (w/v) N-lauroylsarcosine and 1%(v/v) 2-mercaptoethanol were added. TheN-lauroylsarcosine/2-mercaptoethanol supernatant was incubated for 2 hat 37° C. while shaking on an orbital shaker. Subsequently,ultracentrifugation at 108 000×g for 1.5 h at room temperature enrichedaggregated tau in the pellet. Supernatant was removed and the pellet wascarefully rinsed twice with a small amount of TBS (50 mM Tris, 150 mMNaCl, pH 7.4). Finally, the aggregated tau pellet was recovered in TBSand resuspended to ensure sample homogeneity. Small aliquots were storedat −80° C.

Enriched aggregated β-amyloid preps were prepared from frozen human ADbrain samples (10 g—occipital cortex with high amyloid plaques load)that were homogenized with 7-fold vol of cold homogenization buffer (250mM sucrose, 20 mM Tris base, 1 mM EDTA, 1 mM EGTA and PhosSTOPphosphatase and cOmplete EDTA-free protease inhibitor) on ice. Aftercentrifugation at 27 000×g for 20 min at 4° C. cell debris was removed.Supernatant containing amyloid plaques was aliquoted and stored at −80°C.

In Vitro Competitive Radioligand Binding Assays

The competitive radioligand binding assays measure the binding of aradiolabeled reference ligand in the presence of a dose responseconcentration range of test compounds.

Briefly, aggregated tau preps were diluted to 100 μg protein/ml in PBSbuffer with 5% ethanol. In a 96-well format, ³H-T808 (specific activity;32.97 Ci/mmol) was added at a final concentration of 10 nM to increasingamounts of test compound in the presence of 20 μg protein of aggregatedtau prep. Nonspecific binding was defined as the number of countsremaining in the presence of 50 μM Thioflavin T (common beta sheetbinder). After 2 h incubation at room temperature, the unbound ligand isremoved by filtration of the binding mixtures over GF/B glass filtersusing a Filtermate 96 harvester instrument (Perkin Elmer, Zaventem,Belgium). The filters were washed three times with PBS buffer containing20% ethanol. After overnight drying of the filter plate, Microscint Oliquid (Perkin Elmer) was added and the amount of radiolabeled ligandbound to the fibrils is measured by liquid scintillation counting in aTopcount instrument (Packard Instrument Company, Connecticut, USA).

Values for half-maximal inhibitory concentration (IC₅₀) were determinedfrom displacement curves of at least two independent experiments usingGraphPad Prism software (GraphPad Software, San Diego, Calif.).

To determine compound binding to aggregated β-amyloid a similar assaywas put in place but with some minor modifications. Briefly, amyloidpreps were diluted to 150 μg protein/ml in 50 mM Tris with 0.1% BSA and5% ethanol. ³H-AV-45 (florbetapir—specific activity; 45.95 Ci/mmol) wasadded at a final concentration of 10 nM to increasing amounts of testcompound in the presence of 30 μg protein of amyloid plaques prep.Nonspecific binding was determined in the presence of 500 μM ThioflavinT. After 150 min incubation at room temperature, the binding mixtureswere filtered over GF/B glass filters. The filters were washed threetimes with PBS buffer containing 20% ethanol. Subsequent steps wereidentical to those described for the aggregated tau preps.

[¹⁹F]-Co. No. 1 showed potent binding (pIC₅₀ 8.24, corresponding to aK_(i) of 2.4 nM) to extracted human tau using [³H]-T808 and weak bindingto extracted human amyloid-beta aggregates (pIC₅₀ 5.18, corresponding toa K_(i) of 3.2 μM) using [³H]-AV-45 in this radiolabel displacementassay.

Immunohistochemistry (IHC): M&M Human Brain

Human AD brain blocks (Braak stage V-VI) were snap-frozen, sliced with acryostat (20 μm thickness) and stored at −80° C. until used forimmunohistochemistry. Slices were dried, fixed in formalin and incubatedwith hydrogen peroxide (DAKO, S2023) for 5 minutes and blocking reagent(PBS1×+0.05% Triton X-100) during 1 hour. Anti-amyloid or anti-tauantibody [(4G8, Covance, SIG-38220), 1/500 dilution in antibody diluentwith background reducing components (DAKO, S3022) or (AT8 (Biema et al.,EMBO J. 1992, 11(4): 1593-7), in-house, 1 mg/ml stock concentration),0.2 μg/mL in antibody diluent with background reducing components (DAKO,S3022)], was applied to the slices for 1 hour. The striatal tissue wasimmunolabeled with anti-MAO-B and anti-MAO-A antibodies (1:100, ThermoFisher) to confirm MAO expression.

After extensive washing, slides were incubated with HRP-conjugatedanti-mouse secondary antibody (Envision, DAKO, K4000), followed bychromogenic DAB labelling (DAKO, K3468). After counterstaining withhematoxylin, slices were dehydrated and mounted with organic mountingmedium (Vectamount, Vector labs, H-5000). FIG. 1c shows β-amyloidpathology in AD brain as detected with 4G8 IHC and FIG. 1b shows taupathology in AD brain as detected with AT8 IHC.

Autoradiography Studies

a) Air-dried frozen, 10-μm-thick slices of the visual cortex of anAD-patient (68-year old female with Braak stage V-VI) were incubated for60 min with [¹⁸F]Co. No. 1 (7.4 kBq/500 μL per slice) and subsequentlywashed with mixtures of PBS and ethanol as described elsewhere (Xia C.F., Arteaga J., Chen G., et al. Alzheimer's Dement. 2013, 1-11). Toassess specificity of binding, slices were incubated with tracer in thepresence of 1 μM of authentic T808 or Co. No. 1. After drying, sliceswere exposed to a phosphor storage screen (super-resolution screen,Perkin Elmer). Screens were read in a Cyclone Plus system (Perkin Elmer,Waltham, Mass., U.S.A.) and analyzed using Optiquant software. Resultsare expressed as digital light units per square mm (DLU/mm²). AdjacentAD slices were immunostained with anti-tau (AT8) and anti-Aβ antibodies(4G8), to correlate with [¹⁸F]Co. No. 1 binding.

Digital autoradiography with [¹⁸F]Co. No. 1 on human AD-slices showedhigh and selective binding to cortical tau-rich regions (FIG. 1, A).Immunohistochemistry with tau and Aβ antibodies, performed on adjacentslices, identified numerous NFT and neuritic plaque deposits, confirmingco-localization of tracer binding with NFTs (FIG. 1, respectively B andC). To assess the specificity of the tracer binding to these NFTs,blocking studies with authentic reference compound Co. No. 1 and thestructurally unrelated reference compound T808 were performed (FIG. 2).Self-block with cold Co. No. 1 resulted in 99% inhibition, whichdemonstrates that binding of [¹⁸F]Co. No. 1 is specific. Binding of[¹⁸F]Co. No. 1 was reduced with 99% in the presence of 1 μM T808,indicating tau-specific binding, since T808 was reported with highaffinity (K_(D)=22 nM) for aggregated tau and high selective for tauover Aβ aggregates (27-fold) (Zhang W., Arteaga J., Cashion D. K. et al.J Alzheimers Dis. 2012, 31(3), 601-612).

b) [³H]-Co. No. 1 binds to frozen slices of AD brain containing tau andAβ pathology, but does not bind tau pathology negative AD tissuecontaining only Aβ plaques (FIG. 3D, E). The tracer was tested at 10 nMand higher concentrations were not investigated in this study.Importantly, [³H]-Co. No. 1 also shows no binding to MAO-rich humantissue (striatum) (FIG. 3A). These data support that our compound isselective for tau pathology over Aβ pathology and MAO.

In comparison, the tau tracer [³H]-THK-5351 and the Aβ tracer [³H]-AV-45were used as THK-5351 is known to also bind MAO. These experiments showthat 10 nM of [³H]-THK-5351 binds to MAO-rich human tissue and thisbinding can be self-blocked by 10 μM THK-5351 (cold), but also by 10 μMCo. No. 1 (cold) (FIG. 3F-H). This indicates that Co. No. 1 at higherconcentrations can bind MAO.

[³H]-THK5351 also binds to AD tissue containing only Aβ plaques (taunegative) and thus confirms the low selectivity of THK-5351 (FIG. 3I,J).

[³H]-AV-45 (selective for Aβ plaques) binds both tau and Aβ pathology(Aβ+/tau+, Tissue #28391) and Aβ pathology only (Aβ+/tau−, Tissue#92/050) human tissues as expected (FIG. 3L-M). As a control for thehuman AD tissues (Tissue #28391: FIG. 4, Tissue #92/050: FIG. 5) andstriatal tissue (FIG. 6) used in this study immunohistochemistry wasperformed. The human AD tissues (#28391; #92/050) were immunolabeledwith 4G8 (anti-amyloid antibody) and AT8 (anti-tau antibody) and clearlydemonstrate that slices from #28391 are Aβ+/tau+, whereas slices from#92/050 are Aβ+/tau− (FIGS. 4 and 5). Furthermore, MAO-B and MAO-Aexpression in the human striatal tissue (#S96/037) was confirmed usingimmunohistochemistry (FIG. 6). These data support the conclusions fromthe in vitro binding observed in FIG. 3.

c) Binding of [¹⁸F] Co. No. 1 to human AD tissue was assessed in thepresence of reported MAO binders. As can be seen in FIG. 7, 0.2 mCi/mLof [¹⁸F] Co. No. 1 showed strong binding to tau pathology in these humanAD slices (FIG. 7A). This binding pattern was maintained when the samplewas incubated with 10 μM of clorgiline, a strong irreversible MAO binderwith moderate selectivity for the A-subtype (FIG. 7B), or 10 μMselegiline (=deprenyl), a strong irreversible MAO-B binder (FIG. 7C).The binding of [¹⁸F] Co. No. 1 could be entirely blocked with 10 μM ofauthentic Co. No. 1 (FIG. 7D). Adjacent AD slices were immunostainedwith anti-tau (AT8) and anti-Aβ (4G8) antibodies to correlate with[¹⁸F]Co. No. 1 binding (FIG. 8).

MicroPET Imaging Studies

Wistar Rats

A dynamic 120-min μPET scan with [¹⁸F]Co. No. 1 or [¹⁸F]T807 wasacquired on a Focus 220 μPET scanner (Concorde Microsystems, Knoxville,Tenn., U.S.A.) on three female Wistar rats simultaneously. The rats werekept under gas anaesthesia during the whole procedure (2.5% isofluranein O₂ at 1 L/min flow rate). Scans were acquired in list mode andacquisition data were Fourier rebinned in 24 time frames (4×15 s, 4×60s, 5×180 s, 8×300 s, 3×600 s). Data, which were 3D maximum a posteriori(3D-MAP) reconstructed, were manually aligned with a rat brain ¹⁸F-FDGtemplate in Paxinos coordinates using an affine transformation, to allowpredefined volumes of interest (VOIs) analysis (Casteels C., et al. J.Nucl. Med. 2006, 47, 1858-1866). Time-activity curves (TACs) of thewhole brain were generated using VOIs with PMOD software (v 3.2, PMODTechnologies, Zürich, Switzerland). Radioactivity concentration in thebrain was expressed as standardized uptake value (SUV, calculated as(radioactivity in Bq in brain/mL)/(total injected dose (Bq)/body weightin g)) as a function of time after tracer injection. Scans were startedimmediately after IV injection of about 50 MBq radiotracer (n=3/tracer).For pre-treatment and displacement studies, cold reference compound Co.No. 1 or T807 was dissolved in a mixture of 5% DMSO, 5% Tween 80 and 40%(2-hydroxypropyl)-β-cyclodextrin, filtered through a 0.22-μm membranefilter (Millex-GV, Millipore) prior to injection. Pre-treatment (n=1)was done by subcutaneous (SC) injection of 10 mg/kg of Co. No. 1 orT807, 60 min prior to radiotracer injection. Displacement (n=1) wasperformed by IV injection of 1 mg/kg Co. No. 1 or T807 30 min afterradiotracer injection. μPET images were compared to a baseline scan(n=1), acquired in a non-treated rat.

Results of the 120-min baseline, pretreatment and chase study of[¹⁸F]Co. No. 1 and benchmark compound [¹⁸F]T807 are shown in FIGS. 9-11(time activity curves, TACs and % SUV_(max)). TACs of the baseline scansof [¹⁸F]Co. No. 1 (FIG. 9) showed high initial brain uptake with a highintensity SUV value in the brain of ˜2.0, compared to ˜1.8 for [¹⁸F]T807(FIG. 10). Bone uptake was observed at later time points for bothcompounds. [¹⁸F]Co. No. 1 had a faster brain wash-out rate (SUV value of0.2 at 60 min p.i.) compared to [¹⁸F]T807 (SUV value of 0.4 at 60 minp.i.) as shown in the % SUV_(max) curves (FIG. 11). No self-blocking orself-chase effect was observed for [¹⁸F]Co. No. 1 indicating absence ofspecific not tau related binding in brain (FIG. 9). Lower brain uptakeduring the baseline scan of [¹⁸F]T807, compared to the pre-treatmentstudy was recorded, probably caused by saturation of metabolic enzymesand/or plasma proteins (FIG. 10). A similar effect was seen 40 min p.i.in the chase study.

MicroPET Imaging Studies

Rhesus Monkey

A dynamic 120-min PET scan with [¹⁸F]Co. No. 1 or [¹⁸F]T807 wasperformed with a Focus 220 μPET scanner on a rhesus monkey (6 y-old maleMacaca mulatta, 7.6 kg), that was sedated with ketamine (Ketalar®) andxylazine (Rompun®) via intramuscular (IM) injection. During scanning themonkey received repeatedly an additional dose of ketamine/xylazine viaIV injection. 02 saturation in blood, breathing frequency and heartbeatfrequency were monitored during the entire experiment. The head of theanimal was placed central in the field of view of the CμPET scanner.Scans were acquired in list mode and Fourier rebinned in 24 time frames(4×15 s, 4×60 s, 5×180 s, 8×300 s, 3×600 s). Data were reconstructedusing a 3D maximum a posteriori (3D-MAP) iterative reconstruction. TACsof the whole brain were generated using VOIs with PMOD software.Radioactivity concentration in the brain is expressed as SUV as afunction of time after tracer injection. Scans were started immediatelyafter IV injection of 185 MBq of [¹⁸F]Co. No. 1 or [¹⁸F]T807 via thevena saphena of the right leg. For the pre-treatment study, coldreference compound Co. No. 1 was dissolved in a mixture of 10% DMSO and40% (2-hydroxypropyl)-β-cyclodextrin, filtered through a 0.22-μmmembrane filter (Millex-GV, Millipore) prior to injection. Pre-treatment(n=1) was done by IV co-injection of 1 mg/kg of cold Co. No. 1 andradiotracer solution. μPET images were compared to a baseline scan(n=1), acquired in the non-treated monkey.

Results of the 120-min baseline scan of [¹⁸F]Co. No. 1 and [¹⁸F]T807 areshown in FIGS. 12-14 (TACs and % SUV_(max)). TACs of the baseline scanof [¹⁸F]Co. No. 1 in the brain show high initial brain uptake (SUV valueof ˜5.4 in the total brain) with a rapid wash-out (FIG. 12). TACs of thebaseline scan of [¹⁸F]T807 in the brain show a slower initial brainuptake (SUV value of ˜1.3) and wash-out (FIG. 13). A homogeneousdistribution of [¹⁸F]Co. No. 1 was recorded in all observed brainregions, with no increased uptake in the corpus callosum. TACs at theside of the skull show that there was no free ¹⁸F-fluoride bone uptakeobserved 120 min p.i., since the SUV signal did not increase as afunction of time.

The invention claimed is:
 1. A compound according to Formula (I′)

wherein R² is methyl, and either R¹ is ¹⁸F and R³ is H, or R¹ is H and R³ is ¹⁸F; or R¹ and R³ are both H, and R² is selected from the group consisting of —C₁₋₄alkyl-¹⁸F, —OC₁₋₄alkyl-¹⁸F, and —NR⁴—C₁₋₄alkyl-¹⁸F, wherein R⁴ is H or methyl; or a pharmaceutically acceptable salt or a solvate thereof.
 2. The compound according to claim 1, wherein the compound is

or a pharmaceutically acceptable salt or a solvate thereof.
 3. A pharmaceutical composition comprising the compound of claim 1, or a pharmaceutically acceptable salt or a solvate thereof, and a pharmaceutically acceptable carrier or diluent.
 4. The pharmaceutical composition according to claim 3, wherein the composition is a sterile solution.
 5. The compound of claim 1, or a pharmaceutical composition thereof for use in binding and imaging tau aggregates.
 6. The compound of claim 1, or a pharmaceutical composition thereof for use in diagnostic imaging of tau aggregates in the brain of a subject.
 7. The compound of claim 1, or a pharmaceutical composition thereof for use in binding and imaging tau aggregates in a patient suffering from a tauopathy.
 8. A method of binding and imaging tau aggregates in a subject in need thereof, the method comprising administering to the subject the compound of claim 1 or a pharmaceutical composition thereof.
 9. A compound having the formula [¹⁹F]-(I)

wherein R² is methyl, and either R¹ is F and R³ is H, or R¹ is H and R³ is F; or R¹ and R³ are both H, and R² is selected from the group consisting of —C₁₋₄alkyl-F, —OC₁₋₄alkyl-F, and —NR⁴—C₁₋₄alkyl-F, wherein R⁴ is H or methyl; or a pharmaceutically acceptable salt or a solvate thereof.
 10. A method of imaging tau aggregates in a patient comprising administering the compound of claim 1 or a pharmaceutical composition thereof to the patient.
 11. The method of claim 10, wherein the imaging is diagnostic imaging of tau aggregates in the brain of the patient.
 12. The method of claim 11, wherein the patient is suspected of suffering from a tauopathy.
 13. The method of claim 12, wherein the tauopathy is Alzheimer's Disease.
 14. The method of claim 10, wherein the patient is suffering from a tauopathy.
 15. The method of claim 14, wherein the tauopathy is Alzheimer's Disease.
 16. The method of claim 10, wherein the imaging is positron-emission tomography (PET) imaging. 